Pegylated, Extended Insulins

ABSTRACT

PEGylated, extended insulins are insulins which, compared with human insulin, has one or more extensions extended from the A1, B1, A21 and/or B30 position(s), said extension(s) consist(s) of amino acid residue(s) and wherein a PEG moiety, via a linker, is attached to one or more of the amino acid residues in the extension(s). PEG is polyethyleneglycol. Such PEGylated, extended insulins have higher bioavailability and a longer time-action profile than regulär insulin and are in particular suited for pulmonary administration and can, conveniently, be used to treat diabetes.

FIELD OF THIS INVENTION

The present invention is related to PEGylated, extended insulins whichhave insulin activity and can be used for the treatment of diabetes. ThePEGylated, extended insulins have higher bioavailability and a longertime-action profile than regular insulin and are in particular suitedfor pulmonary administration. They will also have a high physicalstability and a low tendency to fibrillation and will be soluble atneutral pH. This invention is also related to pharmaceuticalcompositions containing the PEGylated, extended insulins.

BACKGROUND OF THIS INVENTION

The inherited physical and chemical stability of the insulin molecule isa basic condition for insulin therapy of diabetes mellitus. These basicproperties are fundamental for insulin formulation and for applicableinsulin administration methods, as well as for shelf-life and storageconditions of pharmaceutical preparations. Use of solutions inadministration of insulin exposes the molecule to a combination offactors, e.g., elevated temperature, variable air-liquid-solidinterphases as well as shear forces, which may result in irreversibleconformation changes, e.g., fibrillation.

Unfortunately, many diabetics are unwilling to undertake intensivetherapy due to the discomfort associated with the many injectionsrequired to maintain close control of glucose levels. This type oftherapy can be both psychologically and physically painful. Upon oraladministration, insulin is rapidly degraded in the gastro intestinaltract and is not absorbed into the blood stream. Therefore, manyinvestigators have studied alternate routes for administering insulin,such as oral, rectal, transdermal, and nasal routes. Thus far, however,these routes of administration have not resulted in effective insulinabsorption.

Efficient pulmonary delivery of a protein is dependent on the ability todeliver the protein to the deep lung alveolar epithelium. Proteins thatare deposited in the upper airway epithelium are not absorbed to asignificant extent. This is due to the overlying mucus which isapproximately 30-40 μm thick and acts as a barrier to absorption. Inaddition, proteins deposited on this epithelium are cleared bymucociliary transport up the airways and then eliminated via thegastrointestinal tract. This mechanism also contributes substantially tothe low absorption of some protein particles. The extent to whichproteins are not absorbed and instead eliminated by these routes dependson their solubility, their size, as well as other less understoodcharacteristics.

It is, however, well recognised that the properties of peptides can beenhanced by grafting organic chain-like molecules onto them. Suchgrafting can improve pharmaceutical properties such as half life inserum, stability against proteolytical degradation and reducedimmunogenicity.

The organic chain-like molecules often used to enhance properties arepolyethylene glycolbased chains, i.e., chains that are based on therepeating unit —CH₂CH₂O—. Hereinafter, the abbreviation “PEG” is usedfor polyethyleneglycol.

Classical PEG technology takes advantage of providing polypeptides withincreased size (Stoke radius) by attaching a soluble organic molecule tothe polypeptide (Kochendoerfer, G., et al., Science (299) 884 et seq.,2003). This technology leads to reduced clearance in man and animals ofa hormone polypeptide compared to the native polypeptide. However, thistechnique is often hampered by reduced potency of the hormonepolypeptides subjected to this technique (Hinds, K., et al.,Bioconjugate Chem. (11), 195-201, 2000).

Insulin compositions for pulmonary administration comprising a conjugateof two-chain insulin covalently coupled to one or more molecules ofnon-naturally hydrophilic polymers including polyalkylene glycols andmethods for their preparation are disclosed in WO 02/094200 and WO03/022996.

OBJECTS OF THIS INVENTION

There is still a need for insulins having a more prolonged profile ofaction than the insulin derivatives known up till now and which at thesame time are soluble at physiological pH values and have a potencywhich is comparable to that of human insulin. Furthermore, there is needfor further insulin formulations which are well suited for pulmonaryapplication.

An aspect of this invention deals with furnishing of a medicament whichcan conveniently be administered pulmonary to treat diabetic patients.

Another aspect of this invention deals with the furnishing of amedicament which can conveniently be administered pulmonary to treatdiabetic patients and to reduce the risk of some of or all of the latecomplications often associated with diabetes.

Another aspect of this invention deals with the furnishing of amedicament which can conveniently be administered pulmonary to treatdiabetic patients and which is more convenient to use for many patientsthat the use of injections.

Another aspect of this invention deals with the furnishing of amedicament which can conveniently be administered pulmonary to treatdiabetic patients and which has a sufficient chemical stability.

Another aspect of this invention deals with the furnishing of amedicament which can conveniently be administered pulmonary to treatdiabetic patients and which has a sufficient physical stability.

Another aspect of this invention deals with the furnishing of amedicament having a sufficiently high insulin receptor affinity.

The object of this invention is to overcome or ameliorate at least oneof the disadvantages of the prior art, or to provide a usefulalternative.

DEFINITIONS

Insulin is a polypeptide hormone secreted by β-cells of the pancreas andconsists of two polypeptide chains designated the A and B chains whichare linked together by two inter-chain disulphide bridges. The hormoneis synthesized as a single-chain precursor proinsulin (preproinsulin)consisting of a prepeptide of 24 amino acid followed by proinsulincontaining 86 amino acids in the configuration:prepeptide-B-Arg-Arg-C-Lys-Arg-A, in which C is a connecting peptide of31 amino acids, and A and B are the A and B chains, respectively, ofinsulin. Arg-Arg and Lys-Arg are cleavage sites for cleavage of theconnecting peptide between the A and B chains to form the two-chaininsulin molecule. Insulin is essential in maintaining normal metabolicregulation.

Herein, the term insulin covers natural occurring insulins, e.g., humaninsulin, as well as insulin analogues thereof.

Herein the term amino acid residue covers an amino acid from which ahydrogen atom has been removed from an amino group and/or a hydroxygroup has been removed from a carboxy group and/or a hydrogen atom hasbeen removed from a mercapto group. Imprecise, an amino acid residue maybe designated an amino acid.

Herein, the term insulin analogue covers a polypeptide which has amolecular structure which formally can be derived from the structure ofa naturally occurring insulin, e.g., human insulin, by deleting and/orsubstituting (replacing) one or more amino acid residue occurring in thenatural insulin and/or by adding one or more amino acid residue. Theadded and/or substituted amino acid residues can either be codable aminoacid residues or other naturally occurring amino acid residues or purelysynthetic amino acid residues.

Herein, the term extended insulin covers an insulin analogue whereinthere (compared with human insulin) is added one or more amino acidresidue either C- or N-terminally to the A- or B-chain of insulin. Forexample, the A chain may be extended at its C-terminal end, e.g., by 1,2, 3 or 4 amino acid residues (compared with human insulin) whichextensions are denoted A22, A23, A24 and A25, respectively. For example,when the amino acid residue in position A23 is PEGylated, then the aminoacid in position A22 may be any amino acid residue except Cys and Lys,and so forth. For example, the A chain may be extended at its N-terminalend, e.g., by 1, 2, 3 or 4 amino acid residues (compared with humaninsulin) which extensions are denoted A-1, A-2, A-3 and A-4,respectively. For example, when the amino acid residue in position A-2is PEGylated, then the amino acid in position A-1 may be any amino acidresidue except Cys and Lys, and so forth. Even though the extendedinsulin has an extension at one of the four termini, there may bedeletions at other positions in said extended insulin. Similarly as withhuman insulin, the extended insulin consists of two chains, i.e., the Achain and B chain. In the extended insulin, there are six cysteineresidues, two of which are present in the A chain forming an intra-chaindisulphide bridge (corresponding to A6 and A11 in human insulin) andfour of which form two inter-chain disulphide bridges (corresponding topositions A7, A20, B7 and B19 in human insulin). Herein, the lastmentioned four cysteine residues are designated inter-chain cysteineresidues. In each chain (A and B chain), one of the inter-chain cysteineresidues is closest to the N terminal end of each chain and the otherinter-chain cysteine residues is closest to the C terminal end of eachchain and, herein, such inter-chain cysteine residues are designated anN terminal inter-chain cysteine residue and a C terminal inter-chaincysteine residue, respectively. When determining whether an insulinanalogue is an extended insulin, one has to count the number of aminoacid residues present in each chain on the N terminal side of the Nterminal inter-chain cysteine residue and to count the number of aminoacid residues present in each chain on the C terminal side of the Cterminal inter-chain cysteine residue. If one of these numbers (figures)is larger that the corresponding number for human insulin, that insulinis considered an extended insulin. In human insulin, there are six aminoacid residues present on the N terminal side of the N terminalinter-chain cysteine residue in the A chain, one amino acid residuepresent on the C terminal side of the C terminal inter-chain cysteineresidue in the A chain, six amino acid residues present on the Nterminal side of the N terminal inter-chain cysteine residue in the Bchain, and eleven amino acid residues present on the C terminal side ofthe C terminal inter-chain cysteine residue in the B chain.

Herein the term parent insulin means the extended insulin withoutappended PEG moieties.

Herein, the term mutation covers any change in amino acid sequence(substitutions and insertions with codable amino acids as well asdeletions).

Herein, the term analogues of the A chain and analogues of the B chainsof human insulin covers A and B chains of human insulin, respectively,having one or more substitutions, deletions and or extensions(additions) of the A and B amino acid chains, respectively, relative tothe A and B chains, respectively, of human insulin.

Herein terms like A1, A2, A3 etc. indicates the position 1, 2 and 3,respectively, in the A chain of insulin (counted from the N-terminalend). Similarly, terms like B1, B2, B3 etc. indicates the position 1, 2and 3, respectively, in the B chain of insulin (counted from theN-terminal end). Using the one letter codes for amino acids, terms likeA21A, A21G and A21Q designates that the amino acid in the A21 positionis A, G and Q, respectively. Using the three letter codes for aminoacids, the corresponding expressions are AlaA21, GlyA21 and GInA21,respectively.

Herein terms like A-1, B-1, etcetera, indicates the positions of thefirst amino acids N-terminally to the A1 and B1 positions, respectively,and so forth.

Herein terms like desB29 and desB30 indicate an insulin analogue lackingthe B29 or B30 amino acid residue, respectively.

Herein the term single chain insulin covers a polypeptide sequence ofthe general structure BC-A, wherein A is the A chain of human insulin oran analogue thereof, B is the B chain of human insulin or an analoguethereof, and C is a bond or the so-called connecting peptide, e.g., apeptide chain of about 1-35 amino acid residues connecting theC-terminal amino acid residue in the B-chain, e.g., B30, with theN-terminal amino acid residue in the A-chain, e.g., A1. If the B chainis a desB30 chain, the connecting peptide (C) will connect B29 with A1.The single-chain insulin will contain the three, correctly positioneddisulphide bridges as in human insulin, i.e., between Cys^(A7) andCys^(B7), between Cys^(A20) and Cys^(B19) and between Cys^(A6) andCys^(A11).

The term connecting peptide covers a peptide chain which can connect theC-terminal amino acid residue of the B-chain with the N-terminal aminoacid residue of the A-chain in insuin. Herein the expression B′A means asingle chain insulin wherein the connecting peptide does not consist anany amino acids but simply is a bond, i.e. there is a bond between theB-chain C-terminal and the A-chain N-terminal.

With fast acting insulin is meant an insulin having a faster onset ofaction than normal or regular human insulin.

With long acting insulin is meant an insulin having a longer duration ofaction than normal or regular human insulin.

The numbering of the positions in insulin analogues, extended insulinsand A and B chains is done so that the parent compound is human insulinwith the numbering used for it.

The term basal insulin as used herein means an insulin peptide which hasa time-action of more than 8 hours, in particularly of at least 9 hours.Preferably, the basal insulin has a time-action of at least 10 hours.The basal insulin may thus have a time-action in the range from about 8to 24 hours, preferably in the range from about 9 to about 15 hours.

Herein the term linker covers a chemical moiety which connects an —HN—group of the extended insulin with the —O— group of the PEG moiety. Thelinker does not have any influence on the desired action of the finalPEGylated extended insulin, especially it does not have any adverseinfluence.

With “PEG” or polyethylene glycol, as used herein is meant any watersoluble poly(ethylene glycole) or poly(ethylene oxide). The expressionPEG will comprise the structure —(CH₂CH₂O)_(n)—, where n is an integerfrom 2 to about 1000. A commonly used PEG is end-capped PEG, wherein oneend of the PEG termini is end-capped with a relatively inactive groupsuch as alkoxy, while the other end is a hydroxyl group that may befurther modified by linker moieties. An often used capping group ismethoxy and the corresponding end-capped PEG is often denoted mPEG.Hence, mPEG is CH₃O(CH₂CH₂O)_(n)—, where n is an integer from 2 to about1000 sufficient to give the average molecular weight indicated for thewhole PEG moiety, e.g., for mPEG Mw 2,000, n is approximately 44 (anumber that is subject for batch-to-batch variation). The notion PEG isoften used instead of mPEG. “PEG” followed by a number (not being asubscript) indicates a PEG moiety with the approximate molecular weightequal the number. Hence, “PEG2000” is a PEG moiety having an approximatemolecular weight of 2000.

Specific PEG forms of this invention are branched, linear, forked,dumbbell PEGs, and the like and the PEG groups are typicallypolydisperse, possessing a low polydispersity index of less than about1.05. The PEG moieties present in an extended insulin will for a givenmolecular weight typically consist of a range of ethyleneglycol (orethyleneoxide) monomers. For example, a PEG moiety of molecular weight2000 will typically consist of 44±10 monomers, the average being around44 monomers. The molecular weight (and number of monomers) willtypically be subject to some batch-to-batch variation.

Other specific PEG forms are monodisperse that can be branched, linear,forked, or dumbbell shaped as well. Being monodisperse means that thelength (or molecular weight) of the PEG polymer is specifically definedand is not a mixture of various lengths (or molecular weights). Hereinthe notion mdPEG is used to indicate that the mPEG moiety ismonodisperse, using “d” for “discrete”. The number in subscript aftermdPEG, for example “12” in mdPEG₁₂, indicates the number ofethyleneglycol monomers within the monodisperse polymer (oligomer).

The term PEGylation covers modification of insulin by attachment of oneor more PEG moieties via a linker. The PEG moiety can either be attachedby nucleophilic substitution (acylation) on N-terminal alpha-aminogroups or on lysine residue(s) on the gamma-positions, e.g., withOSu-activated esters, or PEG moieties can be attached by reductivealkylation—also on amino groups present in the extended insulinmolecule—using PEG-aldehyde reagents and a reducing agent, such assodium cyanoborohydride, or, alternatively, PEG moieties can be attachedto the sidechain of an unpaired cysteine residue in a Michael additionreaction using eg. PEG maleimide reagents.

By PEGylated, extended insulin having insulin activity is meant aPEGylated, extended insulin with either the ability to lower the bloodglucose in mammalians as measured in a suitable animal model, which maybe a rat, rabbit, or pig model, after suitable administration e.g., byintravenous, subcutaneous, or pulmonary administration, or an insulinreceptor binding affinity.

Herein the term alkyl covers a saturated, branched or straighthydrocarbon group.

Herein the term alkoxy covers the radical “alkyl-O—”. Representativeexamples are methoxy, ethoxy, propoxy (e.g., 1-propoxy and 2-propoxy),butoxy (e.g., 1-butoxy, 2-butoxy and 2-methyl-2-propoxy), pentoxy(1-pentoxy and 2-pentoxy), hexoxy (1-hexoxy and 3-hexoxy), and the like.

Herein the term alkylene covers a saturated, branched or straightbivalent hydrocarbon group having from 1 to 12 carbon atoms.Representative examples include, but are not limited to, methylene,1,2-ethylene, 1,3-propylene, 1,2-propylene, 1,3-butylene, 1,4-butylene,1,4-pentylene, 1,5-pentylene, 1,5-hexylene, 1,6-hexylene, and the like.

By high physical stability is meant a tendency to fibrillation beingless than 50% of that of human insulin. Fibrillation may be described bythe lag time before fibril formation is initiated at a given conditions.

A polypeptide with insulin receptor and IGF-1 receptor affinity is apolypeptide which is capable of interacting with an insulin receptor anda human IGF-1 receptor in a suitable binding assay. Such receptor assaysare well-know within the field and are further described in theexamples. The present PEGylated, extended insulin will not bind to theIGF-1 receptor or will have a rather low affinity to said receptor. Moreprecisely, the PEGylated, extended insulins of this invention will havean affinity towards the IGF-1 receptor of substantially the samemagnitude or less as that of human insulin

The terms treatment and treating as used herein means the management andcare of a patient for the purpose of combating a disease, disorder orcondition. The term is intended to include the delaying of theprogression of the disease, disorder or condition, the alleviation orrelief of symptoms and complications, and/or the cure or elimination ofthe disease, disorder or condition. The patient to be treated ispreferably a mammal, in particular a human being.

The term treatment of a disease as used herein means the management andcare of a patient having developed the disease, condition or disorder.The purpose of treatment is to combat the disease, condition ordisorder. Treatment includes the administration of the active compoundsto eliminate or control the disease, condition or disorder as well as toalleviate the symptoms or complications associated with the disease,condition or disorder.

The term prevention of a disease as used herein is defined as themanagement and care of an individual at risk of developing the diseaseprior to the clinical onset of the disease. The purpose of prevention isto combat the development of the disease, condition or disorder, andincludes the administration of the active compounds to prevent or delaythe onset of the symptoms or complications and to prevent or delay thedevelopment of related diseases, conditions or disorders.

The term effective amount as used herein means a dosage which issufficient in order for the treatment of the patient to be effectivecompared with no treatment.

POT is the Schizosaccharomyces pombe triose phosphate isomerase gene,and TPI1 is the S. cerevisiae triose phosphate isomerase gene.

By a leader is meant an amino acid sequence consisting of a pre-peptide(the signal peptide) and a pro-peptide.

The term signal peptide is understood to mean a pre-peptide which ispresent as an N-terminal sequence on the precursor form of a protein.The function of the signal peptide is to allow the heterologous proteinto facilitate translocation into the endoplasmic reticulum. The signalpeptide is normally cleaved off in the course of this process. Thesignal peptide may be heterologous or homologous to the yeast organismproducing the protein. A number of signal peptides which may be usedwith the DNA construct of this invention including yeast asparticprotease 3 (YAP3) signal peptide or any functional analog (Egel-Mitaniet al. (1990) YEAST 6:127-137 and U.S. Pat. No. 5,726,038) and theα-factor signal of the MFα1 gene (Thorner (1981) in The MolecularBiology of the Yeast Saccharomyces cerevisiae, Strathern et al., eds.,pp 143-180, Cold Spring Harbor Laboratory, NY and U.S. Pat. No.4,870,00.

The term pro-peptide means a polypeptide sequence whose function is toallow the expressed polypeptide to be directed from the endoplasmicreticulum to the Golgi apparatus and further to a secretory vesicle forsecretion into the culture medium (i.e. exportation of the polypeptideacross the cell wall or at least through the cellular membrane into theperiplasmic space of the yeast cell). The pro-peptide may be the yeastα-factor pro-peptide, vide U.S. Pat. Nos. 4,546,082 and 4,870,008.Alternatively, the pro-peptide may be a synthetic pro-peptide, which isto say a pro-peptide not found in nature. Suitable syntheticpro-peptides are those disclosed in U.S. Pat. Nos. 5,395,922; 5,795,746;5,162,498 and WO 98/32867. The pro-peptide will preferably contain anendopeptidase processing site at the C-terminal end, such as a Lys-Argsequence or any functional analogue thereof.

In the present context, the three-letter or one-letter indications ofthe amino acids have been used in their conventional meaning asindicated in the following. Unless indicated explicitly, the amino acidsmentioned herein are L-amino acids. Further, the left and right ends ofan amino acid sequence of a peptide are, respectively, the N- andC-termini, unless otherwise specified.

Abbreviations for Amino Acids

Amino acid Three-letter code One-letter code Glycine Gly G Proline Pro PAlanine Ala A Valine Val V Leucine Leu L Isoleucine Ile I Methionine MetM Cysteine Cys C Phenylalanine Phe F Tyrosine Tyr Y Tryptophan Trp WHistidine His H Lysine Lys K Arginine Arg R Glutamine Gln Q AsparagineAsn N Glutamic Acid Glu E Aspartic Acid Asp D Serine Ser S Threonine ThrTThe amino acids present in the PEGylated insulins of this invention are,preferably, amino acids which can be coded fro by a nucleic acid.

The following abbreviations have been used in the specification andexamples: Da is Dalton (molecular weight), kDa is kilo-Dalton (=1000Da), mPEG-SBA is mPEG-CH₂CH₂CH₂—CO—OSu (N-hydroxysuccinimidyl ester ofmPEG-butanoic acid), mPEG-SMB is mPEG-CH₂CH₂CH(CH₃)—CO—OSu(N-hydroxysuccinimidyl ester of mPEG-α-methylbutanoic acid), mPEG-SPA ismPEG-CH₂CH₂—CO—OSu (N-hydroxysuccinimidyl ester of mPEG-propionic acid),Mw is molecular weight, OSu is1-succinimidyloxy=2,5-dioxopyrrolidin-1-yloxy, R is room temperature, SAis sinapinic acid and Su is 1-succinimidyl=2,5-dioxopyrrolidin-1-yl.

SUMMARY OF THIS INVENTION

In one aspect, this invention is related to a PEGylated insulin analoguewhich, compared with human insulin, has one or more extensions extendedfrom the A1, B1, A21 and/or B30 position(s), said extension(s)consist(s) of amino acid residue(s) and wherein the PEG moiety, via alinker, is attached to one or more of the amino acid residues in theextension(s).

Via a suitable linker group, a PEG group can be attached to sidechain(s) of lysine or cysteine residue(s) when present or attached tothe N-terminal amino group(s) or at both places in the parent insulin.The linker is typically a derivative of a carboxylic acid, where thecarboxylic acid functionality is used for attachment to the parentinsulin via an amide bond. The linker may be an acetic acid moiety withthe linking motif: —CH₂CO—, a propionic acid moiety with the linkingmotif: —CH₂CH₂CO— or —CHCH₃CO—, or a butyric acid moiety with thelinking motif: —CH₂CH₂CH₂CO— or —CH₂CHCH₃CO—. Alternatively, the linkermay be a —CO— group.

Since PEGylation of the lysine group present in position B29 in thehuman insulin B-chain is unwanted, this amino acid residue shall bereplaced by another amino acid residue. Suitable replacement amino acidresidues are Ala, Arg, Gln and His. Furthermore, it is desirable thatthere is no Lys present in any of the positions 1 through 21 in the Achain (A1-A21) and no Lys present in any of the positions 1 through 30in the B chain (B1-B30).

The parent insulin molecule may have a limited number of the naturallyoccurring amino acid residues substituted with other amino acid residuesas explained in the detailed part of the specification.

In one embodiment, this invention relates to a PEGylated, extendedinsulin, wherein the parent insulin analogue deviates from human insulinin one or more of the following deletions or substitutions: E or D inposition A14, Q in position A18, A, G or Q in position A21, G or Q inposition B1 or no amino acid residue in position B1, Q, S or T inposition B3 or no amino acid residue in position B3, Q in position B13,H in position B25 or no amino acid residue in position B25, no aminoacid residue in position B27, D, E or R in position B28, P, Q or R inposition B29 or no amino acid residue in position B29, no amino acidresidue in position B30.

The PEG group may vary in size within a large range as is well knownwithin the art. However, too large PEG groups may interfere in anegative way with the biological activity of the PEGylated, extendedinsulin molecule.

In still a further aspect, this invention is related to pharmaceuticalpreparations comprising the PEGylated, extended insulin of thisinvention and suitable adjuvants and additives such as one or moreagents suitable for stabilization, preservation or isotoni, e.g., zincions, phenol, cresol, a parabene, sodium chloride, glycerol or mannitol.The zinc content of the present formulations may be between 0 and about6 zinc atoms per insulin hexamer. The pH value of the pharmaceuticalpreparation may be between about 4 and about 8.5, between about 4 andabout 5 or between about 6.5 and about 7.5.

In a further embodiment, this invention is related to the use of thePEGylated, extended insulin as a pharmaceutical for the reducing ofblood glucose levels in mammalians, in particularly for the treatment ofdiabetes.

In a further aspect, this invention is related to the use of thePEGylated, extended insulin for the preparation of a pharmaceuticalpreparation for the reducing of blood glucose level in mammalians, inparticularly for the treatment of diabetes.

In a further embodiment, this invention is related to a method ofreducing the blood glucose level in mammalians by administrating atherapeutically active dose of a PEGylated, extended insulin of thisinvention to a patient in need of such treatment.

In a further aspect of this invention, the PEGylated, extended insulinsare administered in combination with one or more further activesubstances in any suitable ratios. Such further active agents may beselected from human insulin, fast acting insulin analogues, antidiabeticagents, antihyperlipidemic agents, antiobesity agents, antihypertensiveagents and agents for the treatment of complications resulting from orassociated with diabetes.

In one embodiment, the two active components are administered as a mixedpharmaceutical preparation. In another embodiment, the two componentsare administered separately either simultaneously or sequentially.

In one embodiment, the PEGylated, extended insulins of this inventionmay be administered together with fast acting human insulin or humaninsulin analogues. Such fast acting insulin analogue may be such whereinthe amino acid residue in position B28 is Asp, Lys, Leu, Val, or Ala andthe amino acid residue in position B29 is Lys or Pro, des(B28-B30),des(B27) or des(B30) human insulin, and an analogue wherein the aminoacid residue in position B3 is Lys and the amino acid residue inposition B29 is Glu or Asp. The PEGylated, extended insulin of thisinvention and the rapid acting human insulin or human insulin analoguecan be mixed in a ratio from about 90/10%; about 70/30% or about 50/50%.

The PEGylated, extended insulins of this invention may also be used oncombination treatment together with an antidiabetic agent.

Antidiabetic agents will include insulin, GLP-1 (1-37) (glucagon likepeptide-1) described in WO 98/08871, WO 99/43706, U.S. Pat. No.5,424,286 and WO 00/09666, GLP-2, exendin-4(1-39), insulinotropicfragments thereof, insulinotropic analogues thereof and insulinotropicderivatives thereof. Insulinotropic fragments of GLP-1 (1-37) areinsulinotropic peptides for which the entire sequence can be found inthe sequence of GLP-1 (1-37) and where at least one terminal amino acidhas been deleted.

The PEGylated, extended insulins of this invention may also be used oncombination treatment together with an oral antidiabetic such as athiazolidindione, metformin and other type 2 diabetic pharmaceuticalpreparation for oral treatment.

Furthermore, the PEGylated, extended insulin of this invention may beadministered in combination with one or more antiobesity agents orappetite regulating agents.

In one embodiment this invention is related to a pulmonal pharmaceuticalpreparation comprising the PEGgylated extended insulin of this inventionand suitable adjuvants and additives such as one or more agents suitablefor stabilization, preservation or isotoni, e.g., zinc ions, phenol,cresol, a parabene, sodium chloride, glycerol, propyleneglycol ormannitol.

It should be understood that any suitable combination of the PEGylated,extended insulins with diet and/or exercise, one or more of theabove-mentioned compounds and optionally one or more other activesubstances are considered to be within the scope of this invention.

DETAILED DESCRIPTION OF THIS INVENTION

The stability and solubility properties of insulin are importantunderlying aspects for current insulin therapy. This invention isaddressed to these issues by providing stable, PEGylated, extendedinsulin analogues wherein the PEGylation in the extension decreasesmolecular flexibility and concomitantly reduce the fibrillationpropensity and limit or modify the pH precipitation zone.

The PEGylated, extended insulins of this invention are in particularlyintended for pulmonal administration due to their relatively highbioavailability compared to, e.g., human insulin. Furthermore, thePEGylated, extended insulins will have a protracted insulin activity.

Because virtually all PEG polymers are mixtures of many large molecules,one must resort to averages to describe molecular weight. Among manypossible ways of reporting averages, three are commonly used: the numberaverage, weight average, and z-average molecular weights. The weightaverage is probably the most useful of the three, because it fairlyaccounts for the contributions of different sized chains to the overallbehaviour of the polymer, and correlates best with most of the physicalproperties of interest.

$\begin{matrix}{{Number}\mspace{20mu} {average}\mspace{20mu} M\; {{W\left( {\overset{\_}{M}}_{n} \right)}.}} & \frac{\Sigma \left( {M_{i}N_{i}} \right)}{\Sigma \left( {M_{i}N_{i}} \right)} \\{\; {{Weight}\mspace{25mu} {average}\mspace{20mu} M\; {{W\left( {\overset{\_}{M}}_{w} \right)}.}}} & \frac{\Sigma \left( {M_{i}^{2}N_{i}} \right)}{\Sigma \left( {M_{i}N_{i}} \right)} \\{Z\mspace{25mu} {average}\mspace{20mu} M\; {{W\left( {\overset{\_}{M}}_{z} \right)}.}} & \frac{\Sigma \left( {M_{i}^{3}N_{i}} \right)}{\Sigma \left( {M_{i}^{2}N_{i}} \right)}\end{matrix}$

where N_(i) is the mole-fraction (or the number-fraction) of moleculeswith molecular weight M_(i) in the polymer mixture. The ratio of M_(w)to M_(n) is known as the polydispersity index (PDI), and provides arough indication of the breadth of the distribution. The PDI approaches1.0 (the lower limit) for special polymers with very narrow MWdistributions.

While lower molecular weight PEG groups may be preferred for increasingbioavailability, high molecular weight PEG chains, e.g., having anaverage molecular weight of 4000-6000 daltons or greater, althoughgenerally found to decrease the bioactivity of the insulin molecule, maybe preferred for increasing half-life, e.g., in the case of formulationsfor pulmonary administration.

The PEG groups of this invention will typically comprise a number of(—OCH₂CH₂—) subunits.

The PEG groups of the invention will for a given molecular weighttypically consist of a range of ethyleneglycol (or ethyleneoxide)monomers. For example, a PEG group of molecular weight 2000 dalton willtypically consist of 43±10 monomers, the average being around 43-44monomers.

The parent insulin molecule which is PEGylated in this invention is anextended insulin molecule, i.e., an insulin molecule having one or moreamino acid residues attached to the N-terminal end of the parent Aand/or B chain, e.g., to A1 and/or B1, and/or attached to the C-terminalend of the parent A and/or B chain, e.g., A21 and/or B30, referring tohuman insulin. Preferably, the extended insulin molecule, i.e., theparent insulin, contains at least 52 amino acid residues.

The PEGgylated extended insulins of this invention may bemono-substituted having only one PEG group attached to a lysine aminoacid residue in the parent insulin molecule or to a N-terminal aminoacid residue. Alternatively, the PEGylated, extended insulins of thisinvention may comprise two, three- or four PEG groups. If the extendedinsulin comprises more than one PEG group, it will typically have thesame PEG moiety attached to each lysine group or to the N-terminal aminoacid residue. However, the individual PEG groups may also vary from eachother in size and length.

For example, an extended insulin having the following deviations ascompared to human insulin: A22K, B29R, desB30 and being PEGylated in thelysine residue in position A22 with mPEG-propionic acid, 2 kDa, e.g.,using mPEG-SPA is named A22K(N^(ε)mPEG2000-propionyl) B29R desB30 humaninsulin. It is obvious that if any of the corresponding other PEGylationreagents (Mw 2000 Da), containing other linkers, e.g. the butyric acidlinkers, were used for preparation of that particular compound, the“exact” name of that particular compound would be different, but thesmall molecular differences will not result in any differences inbiological properties. In this application, the PEGylated extendedinsulins are, to a great extent, named as if the linking moiety is apropionic acid linker, irrespective of the actual linker. In fact,within protein PEGylation literature, it is rarely specified whichlinking groups are used. The important variables are, with respect tobiological properties: Size (in Daltons) and shape of the PEG moiety andposition of the PEG attachment within the protein.

The parent insulins are produced by expressing a DNA sequence encodingthe extended insulin in question in a suitable host cell by well knowntechnique as disclosed in, e.g., U.S. Pat. No. 6,500,645. The parentinsulin is either expressed directly or as a precursor molecule whichhas an N-terminal extension on the B-chain. This N-terminal extensionmay have the function of increasing the yield of the directly expressedproduct and may be of up to 15 amino acid residues long. The N-terminalextension is to be cleaved of in vitro after isolation from the culturebroth and will therefore have a cleavage site next to B1. N-terminalextensions of the type suitable in this invention are disclosed in U.S.Pat. No. 5,395,922, and European Patent No. 765,395A.

The polynucleotide sequence coding for the parent insulin may beprepared synthetically by established standard methods, e.g., thephosphoamidite method described by Beaucage et al. (1981) TetrahedronLetters 22:1859-1869, or the method described by Matthes et al. (1984)EMBO Journal 3: 801-805. According to the phosphoamidite method,oligonucleotides are synthesized, e.g., in an automatic DNA synthesizer,purified, duplexed and ligated to form the synthetic DNA construct. Acurrently preferred way of preparing the DNA construct is by polymerasechain reaction (PCR).

The polynucleotide sequences may also be of mixed genomic, cDNA, andsynthetic origin. For example, a genomic or cDNA sequence encoding aleader peptide may be joined to a genomic or cDNA sequence encoding theA and B chains, after which the DNA sequence may be modified at a siteby inserting synthetic oligonucleotides encoding the desired amino acidsequence for homologous recombination in accordance with well-knownprocedures or preferably generating the desired sequence by PCR usingsuitable oligonucleotides.

The recombinant method will typically make use of a vector which iscapable of replicating in the selected microorganism or host cell andwhich carries a polynucleotide sequence encoding the parent insulin. Therecombinant vector may be an autonomously replicating vector, i.e., avector which exists as an extra-chromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g., a plasmid, anextra-chromosomal element, a mini-chromosome, or an artificialchromosome. The vector may contain any means for assuringself-replication. Alternatively, the vector may be one which, whenintroduced into the host cell, is integrated into the genome andreplicated together with the chromosome(s) into which it has beenintegrated. Furthermore, a single vector or plasmid or two or morevectors or plasmids which together contain the total DNA to beintroduced into the genome of the host cell, or a transposon may beused. The vector may be linear or closed circular plasmids and willpreferably contain an element(s) that permits stable integration of thevector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

The recombinant expression vector is capable of replicating in yeast.Examples of sequences which enable the vector to replicate in yeast arethe yeast plasmid 2 μm replication genes REP 1-3 and origin ofreplication.

The vector may contain one or more selectable markers which permit easyselection of trans-formed cells. A selectable marker is a gene theproduct of which provides for biocide or viral resistance, resistance toheavy metals, prototrophy to auxotrophs, and the like. Examples ofbacterial selectable markers are the dal genes from Bacillus subtilis orBacillus lichenifonnis, or markers which confer antibiotic resistancesuch as ampicillin, kanamycin, chloramphenicol or tetracyclineresistance. Selectable markers for use in a filamentous fungal host cellinclude amdS (acetamidase), argB (or nithine carbamoyltransferase), pyrG(orotidine-5′-phosphate decarboxylase) and trpC (anthranilate synthase.Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3,TRP1, and URA3. A well suited selectable marker for yeast is theSchizosaccharomyces pompe TPI gene (Russell (1985) Gene 40:125-130).

In the vector, the polynucleotide sequence is operably connected to asuitable promoter sequence. The promoter may be any nucleic acidsequence which shows transcriptional activity in the host cell of choiceincluding mutant, truncated, and hybrid promoters, and may be obtainedfrom genes encoding extra-cellular or intra-cellular polypeptides eitherhomologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription in abacterial host cell, are the promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus lichenifonnis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), and Bacilluslichenifonnis penicillinase gene (penP). Examples of suitable promotersfor directing the transcription in a filamentous fungal host cell arepromoters obtained from the genes for Aspergillus oryzae TAKA amylase,Rhizomucor miehei aspartic proteinase, Aspergillus niger neutralalpha-amylase, and Aspergillus niger acid stable alpha-amylase. In ayeast host, useful promoters are the Saccharomyces cerevisiae Mal, TPI,ADH or PGK promoters.

The polynucleotide sequence encoding the parent insulin will alsotypically be operably connected to a suitable terminator. In yeast asuitable terminator is the TPI terminator (Alber et al. (1982) J. Mol.Appl. Genet. 1:419-434).

The procedures used to ligate the polynucleotide sequence encoding theparent insulin, the promoter and the terminator, respectively, and toinsert them into a suitable vector containing the information necessaryfor replication in the selected host, are well known to persons skilledin the art. It will be understood that the vector may be constructedeither by first preparing a DNA construct containing the entire DNAsequence encoding the extended insulins of this invention, andsubsequently inserting this fragment into a suitable expression vector,or by sequentially inserting DNA fragments containing geneticinformation for the individual elements (such as the signal,pro-peptide, connecting peptide, A and B chains) followed by ligation.

The vector comprising the polynucleotide sequence encoding the parentinsulin is introduced into a host cell so that the vector is maintainedas a chromosomal integrant or as a self-replicating extra-chromosomalvector. The term “host cell” encompasses any progeny of a parent cellthat is not identical to the parent cell due to mutations that occurduring replication. The host cell may be a unicellular microorganism,e.g., a prokaryote, or a non-unicellular microorganism, e.g., aeukaryote. Useful unicellular cells are bacterial cells such as grampositive bacteria including, but not limited to, a Bacillus cell,Streptomyces cell, or gram negative bacteria such as E. coli andPseudomonas sp. Eukaryote cells may be mammalian, insect, plant, orfungal cells. In one embodiment, the host cell is a yeast cell. Theyeast organism may be any suitable yeast organism which, on cultivation,produces large amounts of the single chain insulin of the invention.Examples of suitable yeast organisms are strains selected from the yeastspecies Saccharomyces cerevisiae, Saccharomyces kluyveri,Schizosaccharomyces pombe, Sacchoromyces uvarum, Kluyveromyces lactis,Hansenula polymorpha, Pichia pastoris, Pichia methanolica, Pichiakluyveri, Yarrowia lipolytica, Candida sp., Candida utilis, Candidacacaoi, Geotrichum sp., and Geotrichum fermentans.

The transformation of the yeast cells may for instance be effected byprotoplast formation followed by transformation in a manner known perse. The medium used to cultivate the cells may be any conventionalmedium suitable for growing yeast organisms. The secreted extendedinsulin, a significant proportion of which will be present in the mediumin correctly processed form, may be recovered from the medium byconventional procedures including separating the yeast cells from themedium by centrifugation, filtration or catching the insulin precursorby an ion exchange matrix or by a reverse phase absorption matrix,precipitating the proteinaceous components of the supernatant orfiltrate by means of a salt, e.g., ammonium sulphate, followed bypurification by a variety of chromatographic procedures, e.g., ionexchange chromatography, affinity chromatography, or the like.

Pharmaceutical Compositions

The PEGylated, extended insulins of this invention may be administeredsubcutaneously, orally, or pulmonary.

For subcutaneous administration, the PEGylated, extended insulins ofthis invention are formulated analogously with the formulation of knowninsulins. Furthermore, for subcutaneous administration, the PEGylated,extended insulins of this invention are administered analogously withthe administration of known insulins and, generally, the physicians arefamiliar with this procedure.

PEGylated, extended insulins of this invention may be administered byinhalation in a dose effective to increase circulating insulin levelsand/or to lower circulating glucose levels. Such administration can beeffective for treating disorders such as diabetes or hyperglycemia.Achieving effective doses of insulin requires administration of aninhaled dose of more than about 0.5 μg/kg to about 50 μg/kg ofPEGylated, extended insulins of this invention. A therapeuticallyeffective amount can be determined by a knowledgeable practitioner, whowill take into account factors including insulin level, blood glucoselevels, the physical condition of the patient, the patient's pulmonarystatus, or the like.

The PEGylated, extended insulins of this invention may be delivered byinhalation to achieve slow absorption and/or reduced systemicalclearance thereof. Different inhalation devices typically providesimilar pharmacokinetics when similar particle sizes and similar levelsof lung deposition are compared.

The PEGylated, extended insulins of this invention may be delivered byany of a variety of inhalation devices known in the art foradministration of a therapeutic agent by inhalation. These devicesinclude metered dose inhalers, nebulizers, dry powder generators,sprayers, and the like. Preferably, the PEGylated, extended insulins ofthis are delivered by a dry powder inhaler or a sprayer. There are aseveral desirable features of an inhalation device for administeringPEGylated, extended insulins of this invention. For example, delivery bythe inhalation device is advantageously reliable, reproducible, andaccurate. The inhalation device should deliver small particles oraerosols, e.g., less than about 10 μm, for example about 1-5 μm, forgood respirability. Some specific examples of commercially availableinhalation devices suitable for the practice of this invention areCyclohaler, Turbohaler™ (Astra), Rotahaler® (Glaxo), Diskus® (Glaxo),Spiros™ inhaler (Dura), devices marketed by Inhale Therapeutics, AERx™(Aradigm), the Ultravent® nebulizer (Mallinckrodt), the Acorn II®nebulizer (Marquest Medical Products), the Ventolin® metered doseinhaler (Glaxo), the Spinhaler® powder inhaler (Fisons), or the like.

As those skilled in the art will recognize, the formulation ofPEGylated, extended insulins of this invention, the quantity of theformulation delivered and the duration of administration of a singledose depend on the type of inhalation device employed. For some aerosoldelivery systems, such as nebulizers, the frequency of administrationand length of time for which the system is activated will depend mainlyon the concentration of PEGylated, extended insulins in the aerosol. Forexample, shorter periods of administration can be used at higherconcentrations of PEGylated, extended insulins in the nebulizersolution. Devices such as metered dose inhalers can produce higheraerosol concentrations, and can be operated for shorter periods todeliver the desired amount of the PEGylated, extended insulins. Devicessuch as powder inhalers deliver active agent until a given charge ofagent is expelled from the device. In this type of inhaler, the amountof insulin PEGylated, extended insulins of this invention in a givenquantity of the powder determines the dose delivered in a singleadministration.

The particle size of PEGylated, extended insulins of this invention inthe formulation delivered by the inhalation device is critical withrespect to the ability of insulin to make it into the lungs, andpreferably into the lower airways or alveoli. Preferably, the PEGylated,extended insulins of this invention ion is formulated so that at leastabout 10% of the PEGylated, extended insulins delivered is deposited inthe lung, preferably about 10 to about 20%, or more. It is known thatthe maximum efficiency of pulmonary deposition for mouth breathinghumans is obtained with particle sizes of about 2 μm to about 3 μm. Whenparticle sizes are above about 5 μm, pulmonary deposition decreasessubstantially. Particle sizes below about 1 μm cause pulmonarydeposition to decrease, and it becomes difficult to deliver particleswith sufficient mass to be therapeutically effective. Thus, particles ofthe PEGylated, extended insulins delivered by inhalation have a particlesize preferably less than about 10 μm, more preferably in the range ofabout 1 μm to about 5 μm. The formulation of the PEGylated, extendedinsulins is selected to yield the desired particle size in the choseninhalation device.

Advantageously for administration as a dry powder a PEGylated, extendedinsulin of this invention is prepared in a particulate form with aparticle size of less than about 10 μm, preferably about 1 to about 5μm. The preferred particle size is effective for delivery to the alveoliof the patient's lung. Preferably, the dry powder is largely composed ofparticles produced so that a majority of the particles have a size inthe desired range. Advantageously, at least about 50% of the dry powderis made of particles having a diameter less than about 10 μm. Suchformulations can be achieved by spray drying, milling, micronisation, orcritical point condensation of a solution containing the PEGylated,extended insulin of this invention and other desired ingredients. Othermethods also suitable for generating particles useful in the currentinvention are known in the art.

The particles are usually separated from a dry powder formulation in acontainer and then transported into the lung of a patient via a carrierair stream. Typically, in current dry powder inhalers, the force forbreaking up the solid is provided solely by the patient's inhalation. Inanother type of inhaler, air flow generated by the patient's inhalationactivates an impeller motor which deagglomerates the particles.

Formulations of PEGylated, extended insulins of this invention foradministration from a dry powder inhaler typically include a finelydivided dry powder containing the derivative, but the powder can alsoinclude a bulking agent, carrier, excipient, another additive, or thelike. Additives can be included in a dry powder formulation ofPEGylated, extended insulin, e.g., to dilute the powder as required fordelivery from the particular powder inhaler, to facilitate processing ofthe formulation, to provide advantageous powder properties to theformulation, to facilitate dispersion of the powder from the inhalationdevice, to stabilize the formulation (for example, antioxidants orbuffers), to provide taste to the formulation, or the like.Advantageously, the additive does not adversely affect the patient'sairways. The PEGylated, extended insulin can be mixed with an additiveat a molecular level or the solid formulation can include particles ofthe PEGylated, extended insulin mixed with or coated on particles of theadditive. Typical additives include mono-, di-, and polysaccharides;sugar alcohols and other polyols, such as, e.g., lactose, glucose,raffinose, melezitose, lactitol, maltitol, trehalose, sucrose, mannitol,starch, or combinations thereof; surfactants, such as sorbitols,diphosphatidyl choline, or lecithin; or the like. Typically an additive,such as a bulking agent, is present in an amount effective for a purposedescribed above, often at about 50% to about 90% by weight of theformulation. Additional agents known in the art for formulation of aprotein such as insulin analogue protein can also be included in theformulation.

A spray including the PEGylated, extended insulins of this invention canbe produced by forcing a suspension or solution of the PEGylated,extended insulin through a nozzle under pressure. The nozzle size andconfiguration, the applied pressure, and the liquid feed rate can bechosen to achieve the desired output and particle size. An electrospraycan be produced, e.g., by an electric field in connection with acapillary or nozzle feed. Advantageously, particles of insulin conjugatedelivered by a sprayer have a particle size less than about 10 μm,preferably in the range of about 1 μm to about 5 μm.

Formulations of PEGylated, extended insulins of this invention suitablefor use with a sprayer will typically include the PEGylated, extendedinsulins in an aqueous solution at a concentration of from about 1 mg toabout 500 mg of the PEGylated, extended insulin per ml of solution.Depending on the PEGylated insulin chosen and other factors known to themedical advisor, the upper limit may be lower, e.g., 450, 400, 350, 300,250, 200, 150, 120, 100 or 50 mg of the PEGylated insulin per ml ofsolution. The formulation can include agents such as an excipient, abuffer, an isotonicity agent, a preservative, a surfactant, and,preferably, zinc. The formulation can also include an excipient or agentfor stabilization of the PEGylated, extended insulin, such as a buffer,a reducing agent, a bulk protein, or a carbohydrate. Bulk proteinsuseful in formulating insulin conjugates include albumin, protamine, orthe like. Typical carbohydrates useful in formulating the PEGylated,extended insulin include sucrose, mannitol, lactose, trehalose, glucose,or the like. The PEGylated, extended insulins formulation can alsoinclude a surfactant, which can reduce or prevent surface-inducedaggregation of the insulin conjugate caused by atomization of thesolution in forming an aerosol. Various conventional surfactants can beemployed, such as polyoxyethylene fatty acid esters and alcohols, andpolyoxyethylene sorbitol fatty acid esters. Amounts will generally rangebetween about 0.001 and about 4% by weight of the formulation.

Pharmaceutical compositions containing a PEGylated, extended insulin ofthis invention may also be administered parenterally to patients in needof such a treatment. Parenteral administration may be performed bysubcutaneous, intramuscular or intravenous injection by means of asyringe, optionally a pen-like syringe. Alternatively, parenteraladministration can be performed by means of an infusion pump.

Injectable compositions of the PEGylated, extended insulins of thisinvention can be prepared using the conventional techniques of thepharmaceutical industry which involve dissolving and mixing theingredients as appropriate to give the desired end product. Thus,according to one procedure, a PEGylated, extended insulin is dissolvedin an amount of water which is somewhat less than the final volume ofthe composition to be prepared. Zink, an isotonic agent, a preservativeand/or a buffer is/are added as required and the pH value of thesolution is adjusted—if necessary—using an acid, e.g., hydrochloricacid, or a base, e.g., aqueous sodium hydroxide as needed. Finally, thevolume of the solution is adjusted with water to give the desiredconcentration of the ingredients.

In a further embodiment of this invention the buffer is selected fromthe group consisting of sodium acetate, sodium carbonate, citrate,glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogenphosphate, disodium hydrogen phosphate, sodium phosphate, andtris(hydroxymethyl)aminomethan, bicine, tricine, malic acid, succinate,maleic acid, fumaric acid, tartaric acid, aspartic acid or mixturesthereof. Each one of these specific buffers constitutes an alternativeembodiment of this invention.

In a further embodiment of this invention the formulation furthercomprises a pharmaceutically acceptable preservative which may beselected from the group consisting of phenol, o-cresol, m-cresol,p-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate,2-phenoxyethanol, butyl p-hydroxybenzoate, 2-phenylethanol, benzylalcohol, chlorobutanol, and thiomerosal, bronopol, benzoic acid,imidurea, chlorohexidine, sodium dehydroacetate, chlorocresol, ethylp-hydroxybenzoate, benzethonium chloride, chlorphenesine(3-(4-chlorophenoxy)-1,2-propanediol) or mixtures thereof. In a furtherembodiment of this invention the preservative is present in aconcentration from about 0.1 mg/ml to 20 mg/ml. In a further embodimentof this invention the preservative is present in a concentration fromabout 0.1 mg/ml to 5 mg/ml. In a further embodiment of this inventionthe preservative is present in a concentration from about 5 mg/ml to 10mg/ml. In a further embodiment of this invention the preservative ispresent in a concentration from about 10 mg/ml to 20 mg/ml. Each one ofthese specific preservatives constitutes an alternative embodiment ofthis invention. The use of a preservative in pharmaceutical compositionsis well-known to the skilled person. For convenience reference is madeto Remington: The Science and Practice of Pharmacy, 1 gth edition, 1995.

In a further embodiment of this invention, the formulation furthercomprises an isotonic agent which may be selected from the groupconsisting of a salt (e.g., sodium chloride), a sugar or sugar alcohol,an amino acid (for example, L-glycine, L-histidine, arginine, lysine,isoleucine, aspartic acid, tryptophan or threonine), an alditol (e.g.glycerol (glycerine), 1,2-propanediol (propyleneglycol), 1,3-propanediolor 1,3-butanediol), polyethyleneglycol (e.g., PEG400) or mixturesthereof. Any sugar such as mono-, di-, or polysaccharides, orwater-soluble glucans, including for example fructose, glucose, mannose,sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran,pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch andcarboxymethylcellulose-Na may be used. In one embodiment the sugaradditive is sucrose. Sugar alcohol is defined as a C4-C8 hydrocarbonhaving at least one—OH group and includes, e.g., mannitol, sorbitol,inositol, galactitol, dulcitol, xylitol, and arabitol. In one embodimentthe sugar alcohol additive is mannitol. The sugars or sugar alcoholsmentioned above may be used individually or in combination. There is nofixed limit to the amount used, as long as the sugar or sugar alcohol issoluble in the liquid preparation and does not adversely effect thestabilizing effects achieved using the methods of this invention. In oneembodiment, the sugar or sugar alcohol concentration is between about 1mg/ml and about 150 mg/ml. In a further embodiment of this invention theisotonic agent is present in a concentration from about 1 mg/ml to 50mg/ml. In a further embodiment of this invention the isotonic agent ispresent in a concentration from about 1 mg/ml to 7 mg/ml. In a furtherembodiment of this invention the isotonic agent is present in aconcentration from about 8 mg/ml to 24 mg/ml. In a further embodiment ofthis invention the isotonic agent is present in a concentration fromabout 25 mg/ml to 50 mg/ml. Each one of these specific isotonic agentsconstitutes an alternative embodiment of this invention. The use of anisotonic agent in pharmaceutical compositions is well-known to theskilled person. For convenience reference is made to Remington: TheScience and Practice of Pharmacy, 19^(th) edition, 1995.

Typical isotonic agents are sodium chloride, mannitol, dimethyl sulfoneand glycerol and typical preservatives are phenol, m-cresol, methylp-hydroxybenzoate and benzyl alcohol.

Examples of suitable buffers are sodium acetate, glycylglycine, HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and sodiumphosphate.

A composition for nasal administration of a PEGylated, extended insulinsof this invention may, e.g., be prepared as described in European PatentNo. 272097.

Compositions containing PEGylated, extended insulins of this inventioncan be used in the treatment of states which are sensitive to insulin.Thus, they can be used in the treatment of type 1 diabetes, type 2diabetes and hyperglycaemia for example as sometimes seen in seriouslyinjured persons and persons who have undergone major surgery. Theoptimal dose level for any patient will depend on a variety of factorsincluding the efficacy of the specific insulin derivative employed, theage, body weight, physical activity, and diet of the patient, on apossible combination with other drugs, and on the severity of the stateto be treated. It is recommended that the daily dosage of the PEGylated,extended insulin of this invention be determined for each individualpatient by those skilled in the art in a similar way as for knowninsulin compositions.

PREFERRED FEATURES OF THIS INVENTION

To sum up, the features of this invention are as follows:

-   1. A PEGylated insulin analogue which, compared with human insulin,    has one or more PEG-containing extensions extended from the A1, B1,    A21 and/or B30 position(s), said extension(s) consist(s) of amino    acid residue(s) and wherein the PEG moiety, via a linker, is    attached to one or more of the amino acid residues in the    extension(s).-   2. A PEGylated insulin analogue, according to clause 1, wherein only    one of the amino acid residues in one of the extensions carries a    PEG moiety.-   3. A PEGylated insulin analogue, according to clause 1, wherein only    two of the extensions carries a PEG moiety, and, preferably, there    are only two PEG moieties.-   4. A PEGylated insulin analogue, according to clause 1, wherein the    extension carrying a PEG moiety is situated in a position    N-terminally to the A1 position.-   5. A PEGylated insulin analogue, according to clause 1, wherein the    extension carrying a PEG moiety is situated in a position    N-terminally to the B1 position.-   6. A PEGylated insulin analogue, according to clause 1, wherein the    extension carrying a PEG moiety is situated in a position    C-terminally to the A21 position.-   7. A PEGylated insulin analogue, according to clause 1, wherein the    extension carrying a PEG moiety is situated in a position    C-terminally to the B30 position.-   8. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the number of extensions per    insulin molecule is four.-   9. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the number of extensions per    insulin molecule is three.-   10. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the number of extensions per    insulin molecule is two.-   11. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the number of extensions per    insulin molecule is only one.-   12. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in one or more of the following    extensions: G in position A-3, G in position A-2, K or R in position    A-1, G or K in position A22, G or K in position A23, G or K in    position A24, K in position A25, and K in position B31 and, compared    with human insulin, there is, optionally, up to 12 more mutations    among deletion, substitution and addition of an amino acid residue    and, preferably, there are no further mutations in said insulin    analogue.-   13. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the extension consists of one    or more of the following formulae wherein the PEG moiety is attached    to side chain(s) of lysine or cysteine residue(s) when present or to    the N-terminal amino group(s) (or both):    -AA_(x1)K (for C-terminal extensions), wherein X1 is 0, 1, 2, 3, 4,    5, 6, 7, or 8 (and wherein K is lysine),    K-AA_(x2)- (for N-terminal extensions), wherein x2 is 0, 1, 2, 3, 4,    5, 6, 7, or 8 (and wherein K is lysine),    -AA_(x3)C (for C-terminal extensions), wherein x3 is 0, 1, 2, 3, 4,    5, 6, 7, or 8 (and wherein C is cysteine),    C-AA_(x4)- (for N-terminal extensions), wherein x4 is 0, 1, 2, 3, 4,    5, 6, 7, or 8 (and wherein C is cysteine),    AA_(x5)-R_(y)- (for N-terminal extensions), wherein x5 is 0, 1, 2,    3, 4, 5, 6, 7, or 8, and y is 0 or 1 (and wherein R is arginine),    and wherein AA is the residue of any codable amino acid except Lys    and Cys, and, preferably, AA is a peptide chain wherein each of the    codable amino acid residues are the same or different.-   14. A PEGylated insulin analogue, according to any one of the    preceding clauses, wherein AA is a residue of glycine, alanine or    glutamine, and, preferably, AA is a peptide chain wherein each of    the codable amino acid residues are the same or different.-   15. A PEGylated insulin analogue, according to the preceding clause,    wherein AA is a residue of glycine.-   16. A PEGylated insulin analogue, according to the preceding clause,    wherein AA is a residue of alanine.-   17. A PEGylated insulin analogue, according to the preceding clause    but one, wherein AA is a residue of glutamine.-   18. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the extension consists of one    or more of the following formulae wherein the PEG moiety is attached    to side chain(s) of lysine or cysteine residue(s) when present or to    the N-terminal amino group(s) (or both):    -G_(x1)K (for C-terminal extensions), wherein X1 is 0, 1, 2, 3, 4,    5, 6, 7, or 8 (and wherein G and K are glycine and lysine,    respectively),    K-G_(x2)- (for N-terminal extensions), wherein x2 is 0, 1, 2, 3, 4,    5, 6, 7, or 8 (and wherein G and K are glycine and lysine,    respectively),    -G_(x3)C (for C-terminal extensions), wherein x3 is 0, 1, 2, 3, 4,    5, 6, 7, or 8 (and wherein G and C are glycine and cysteine,    respectively),    C-G_(x4)- (for N-terminal extensions), wherein x4 is 0, 1, 2, 3, 4,    5, 6, 7, or 8 (and wherein G and C are glycine and cysteine,    respectively),    G_(x5)-R_(y)- (for N-terminal extensions), wherein x5 is 0, 1, 2, 3,    4, 5, 6, 7, or 8, and y is 0 or 1 (and wherein G and R are glycine    and arginine, respectively).-   19. PEGylated insulin according to anyone of the preceding, possible    clauses wherein the parent insulin, optionally contains one or more    of the following mutations: A14E/D, A18Q, A21G/A/Q, desB1, B1G/Q,    B3Q/S/T, B13Q, desB25, B25H, desB27, B28D/E/R, des B29, B29P/Q/R or    desB30.-   20. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A22K, B29R and desB30 and,    preferably, there are no further mutations in said insulin analogue.-   21. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A22G, A23K, B29R and desB30    and, preferably, there are no further mutations in said insulin    analogue.-   22. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A22G, A23G, A24K, B29R and    desB30 and, preferably, there are no further mutations in said    insulin analogue.-   23. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A22G, A23G, A24G, A25K, B29R    and desB30 and, preferably, there are no further mutations in said    insulin analogue.-   24. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A22K, B3Q, B29R and desB30    and, preferably, there are no further mutations in said insulin    analogue.-   25. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A22K, B3S, B29R and desB30    and, preferably, there are no further mutations in said insulin    analogue.-   26. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A22K, B3T, B29R and desB30    and, preferably, there are no further mutations in said insulin    analogue.-   27. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A22K, B1Q, B29R and desB30    and, preferably, there are no further mutations in said insulin    analogue.-   28. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A18Q, A22K, B29R and desB30    and, preferably, there are no further mutations in said insulin    analogue.-   29. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A22K, B3Q, B29R, desB1 and    desB30 and, preferably, there are no further mutations in said    insulin analogue.-   30. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having B29Q and B31K and, preferably,    there are no further mutations in said insulin analogue.-   31. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A21G, B29Q and B31K and,    preferably, there are no further mutations in said insulin analogue.-   32. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A21A, B29Q and B31K and,    preferably, there are no further mutations in said insulin analogue.-   33. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A21Q, B29Q and B31K and,    preferably, there are no further mutations in said insulin analogue.-   34. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A-1K and desB30 and,    preferably, there are no further mutations in said insulin analogue.-   35. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A-1K, B29R and desB30 and,    preferably, there are no further mutations in said insulin analogue.-   36. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A-3G, A-2G, A-1R and desB30    and, preferably, there are no further mutations in said insulin    analogue.-   37. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A22K, B28E, B29R and desB30    and, preferably, there are no further mutations in said insulin    analogue.-   38. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A22K, B28D, B29R and desB30    and, preferably, there are no further mutations in said insulin    analogue.-   39. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A22K, B28E, B29R, desB27 and    desB30 and, preferably, there are no further mutations in said    insulin analogue.-   40. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having B28E, B29Q and B31K and,    preferably, there are no further mutations in said insulin analogue.-   41. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having B28E, B29Q, B31K and desB27    and, preferably, there are no further mutations in said insulin    analogue.-   42. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A22K, B28R, desB29 and desB30    and, preferably, there are no further mutations in said insulin    analogue.-   43. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having B28R, B29P and B31K and,    preferably, there are no further mutations in said insulin analogue.-   44. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A22K, B3Q, B28E, B29R and    desB30 and, preferably, there are no further mutations in said    insulin analogue.-   45. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A21G, B3Q, B28E, B29Q and B31K    and, preferably, there are no further mutations in said insulin    analogue.-   46. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A22K, B13Q, B29R and desB30    and, preferably, there are no further mutations in said insulin    analogue.-   47. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A22K, B29R, desB1 and desB30    and, preferably, there are no further mutations in said insulin    analogue.-   48. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A14E, A22K, B25H and desB30    and, preferably, there are no further mutations in said insulin    analogue.-   49. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A14E, B25H, B29Q and B31K and,    preferably, there are no further mutations in said insulin analogue.-   50. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A13E, A22K, B25H and desB30    and, preferably, there are no further mutations in said insulin    analogue.-   51. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A21Q, A22K, B29R and desB30    and, preferably, there are no further mutations in said insulin    analogue.-   52. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A21Q, A22G, A23K, B29R and    desB30 and, preferably, there are no further mutations in said    insulin analogue.-   53. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A21Q, A22G, A23G, A24K, B29R    and desB30 and, preferably, there are no further mutations in said    insulin analogue.-   54. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A21Q, A22G, A23G, A24G, A25K,    B29R and desB30 and, preferably, there are no further mutations in    said insulin analogue.-   55. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A21A, A22K, B29R and desB30    and, preferably, there are no further mutations in said insulin    analogue.-   56. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A21A, A22G, A23K, B29R and    desB30 and, preferably, there are no further mutations in said    insulin analogue.-   57. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A21A, A22G, A23G, A24K, B29R    and desB30 and, preferably, there are no further mutations in said    insulin analogue.-   58. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A21A, A22G, A23G, A24G, A25K,    B29R and desB30 and, preferably, there are no further mutations in    said insulin analogue.-   59. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A21G, A22K, B29R and desB30    and, preferably, there are no further mutations in said insulin    analogue.-   60. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A21G, A22G, A23K, B29R and    desB30 and, preferably, there are no further mutations in said    insulin analogue.-   61. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A21G, A22G, A23G, A24K, B29R    and desB30 and, preferably, there are no further mutations in said    insulin analogue.-   62. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A21G, A22G, A23G, A24G, A25K,    B29R and desB30 and, preferably, there are no further mutations in    said insulin analogue.-   63. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A21Q, A22K, B3Q, B29R and    desB30 and, preferably, there are no further mutations in said    insulin analogue.-   64. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A21G, A22K, B3Q, B29R and    desB30 and, preferably, there are no further mutations in said    insulin analogue.-   65. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A21A, A22K, B3Q, B29R and    desB30 and, preferably, there are no further mutations in said    insulin analogue.-   66. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the parent insulin analogue    deviates from human insulin in having A14E, A22K, B25H, B29R and    desB30 and, preferably, there are no further mutations in said    insulin analogue.-   67. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein said PEG containing group is    attached to an —NH— group of a lysine residue and/or to a cysteine    residue present in the extension(s) and/or attached N-terminally to    the extension(s).-   68. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, comprising the moiety —(OCH₂CH₂)_(n)—,    wherein n is in integer in the range from 2 to about 1000,    preferably from 2 to about 500, preferably from 2 to about 250,    preferably from 2 to about 125, preferably from 2 to about 50, and    preferably from 2 to about 25.-   69. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the polyethylene glycol moiety    has a nominal molecular weight in the range from about 200 to about    40,000, preferably from about 200 to about 30,000, preferably from    about 200 to about 20,000, preferably from about 200 to about    10,000, preferably from about 200 to about 5,000, preferably from    about 200 to about 2,000, preferably from about 200 to about 1,000,    and preferably from about 200 to about 750.-   70. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the polyethylene glycol moiety    is monodisperse.-   71. A PEGylated insulin analogue, according to the preceding clause,    wherein the polyethylene glycol moiety has the general formula    —(CH₂CH₂O)_(n)—, wherein n is in an integer which is at least about    6, preferably at least about 10, and not more than about 110,    preferably not more than about 75, and even more preferred n is in    the range from about 6 to about 30, preferably in the range from    about 10 to about 48.-   72. A PEGylated insulin analogue, according to any one of the    preceding possible clauses, wherein the polyethylene glycol moiety    is polydisperse.-   73. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein the polyethylene glycol moiety    is linear, branched, forked or dumbbell shaped.-   74. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, comprising a group of the general    formula -Q¹-(OCH₂CH₂)_(n)—R¹ wherein Q¹ is a linker connecting the    polyethylene glycol moiety to an α- or γ-NH-group of an amino acid    in the extension, preferably via an amide or a carbamate bond, n is    an integer in the range from 2 to about 1000, and R¹ is alkoxy or    hydroxyl, preferably methoxy.-   75. A PEGylated insulin analogue, according to the preceding clause,    wherein n is an integer in the range from 2 to about 500, preferably    from 2 to about 500, preferably from 2 to about 250, preferably from    2 to about 125, preferably from 2 to about 50, and preferably from 2    to about 25.-   76. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein Q¹ is -alkylene-CO—, which is    connected to the —NH— residue of the extended insulin via the    carbonyl group.-   77. A PEGylated insulin analogue, according to the preceding clause,    wherein Q¹ is ethylene carbonyl ((CH₂)₂—CO—), which is connected to    the —NH— residue via the carbonyl group.-   78. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses except the two last, wherein Q¹ is    -alkylene-NHCO-alkylene-CO—, which is connected to the —NH— residue    of the extended insulin via the carbonyl group.-   79. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses except the three last, wherein Q¹ is    —CO-alkylene-CO—.-   80. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses except the four last, wherein Q¹ is    —CO-(4-nitrophenoxy).-   81. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses except the five last, wherein Q¹ is    (-alkylene-NHCO-alkylene-O-alkylene-)_(p)CH_(q)—NHCO-alkylene-(OCH₂CH₂)_(r)—NHCO-alkylene-CO—,    wherein p is 1, 2 or 3, q is 0, 1 or 2, p+q is 3, and r is an    integer in the range from 1 to about 12, which is connected to the    —NH— residue of the extended insulin via the carbonyl group.-   82. A PEGylated insulin analogue, according to any on of the    preceding, possible clauses, wherein Q¹ is —CH₂CO—, —CH₂CH₂CO—,    —CH₂CH₂CH₂CO—, —CH₂CH(CH₃)CO—, —CH₂CH₂CH₂CH₂CO—, —CH₂CH₂CH(CH₃)CO—,    —CH₂CH₂CH₂CH₂CH₂CO—, —CH₂CH₂NH—COCH₂CH₂CO—,    —CH₂CH₂NH—COCH₂CH₂CH₂CO—, —CH₂CH₂CH₂NH—COCH₂CH₂CO—,    —CH₂CH₂CH₂NH—COCH₂CH₂CH₂CO—, —COCH₂CH₂CO—, —COCH₂CH₂CH₂CO—,    —CO-(4-nitrophenoxy),    (—CH₂CH₂NHCOCH₂CH₂O—CH₂)₃CNHCOCH₂CH₂(OCH₂CH₂)₄NHCOCH₂CH₂CO— or    (—CH₂CH₂NHCOCH₂CH₂OCH₂)₃CNH—COCH₂CH₂(OCH₂CH₂)₄NHCOCH₂CH₂CH₂CO—.-   83. A PEGylated insulin analogue, according to any one of the    preceding, possible clauses, wherein R¹ is alkoxy.-   84. A PEGylated insulin analogue, according to the preceding clause,    wherein R¹ is methoxy.-   85. A compound according to any one of the preceding product    clauses, which is any one of the compounds mentioned specifically in    the above specification such as in the specific examples, especially    any one of the examples 1 et seq. above-   86. The use of a compound according to any one of the preceding    product clauses for the preparation of a pharmaceutical composition    for the treatment of diabetes.-   87. The use of a compound according to any one of the preceding    product clauses for the preparation of a pharmaceutical composition    which can be administered pulmonary for the treatment of diabetes.-   88. The use of a compound according to any one of the preceding    product clauses for the preparation of a pharmaceutical composition    which can be administered pulmonary for the treatment of diabetes    and which gives a long acting effect.-   89. The use of a compound according to any one of the preceding    product clauses for the preparation of a powder pharmaceutical    composition which can be administered pulmonary for the treatment of    diabetes.-   90. The use of a compound according to any one of the preceding    product clauses for the preparation of a liquid pharmaceutical    composition which can be administered pulmonary for the treatment of    diabetes.-   91. A method of treatment of diabetes, the method comprising    administering to a subject in need thereof a therapeutically    effective amount of a compound according to any one of the preceding    product clauses.-   92. A composition containing human insulin as well as a PEGylated    insulin analogue according to any one of the preceding clauses.-   93. A composition containing insulin aspart as well as a PEGylated    insulin analogue according to any one of the preceding clauses.-   94. A composition containing insulin Lispro as well as a PEGylated    insulin analogue according to any one of the preceding clauses.-   95. A composition containing insulin Glulisine as well as a    PEGylated insulin analogue according to any one of the preceding    clauses.-   96. Any novel feature or combination of features described herein.

General Comments

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference in theirentirety and to the same extent as if each reference were individuallyand specifically indicated to be incorporated by reference and were setforth in its entirety herein (to the maximum extent permitted by law).

All headings and sub-headings are used herein for convenience only andshould not be construed as limiting this invention in any way.

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate this inventionand does not pose a limitation on the scope of this invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthis invention.

The citation and incorporation of patent documents herein is done forconvenience only and does not reflect any view of the validity,patentability, and/or enforceability of such patent documents. Thementioning herein of references is no admission that they constituteprior art.

Herein, the word “comprise” is to be interpreted broadly meaning“include”, “contain” or “comprehend” (EPO guidelines C 4.13).

This invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw.

Combining one or more of the embodiments described herein, optionallyalso with one or more of the claims below, results in furtherembodiments and this invention relates to all possible combinations ofsaid embodiments and claims.

For the sake of completeness, it is to be noted that this invention doesnot relate to PEGylated insulin analogues wherein the parent insulinanalogue is so-called single-chain insulin (Danish appl. No.: 2005/00400and WO appl. No.: EP2006/060816; our ref.: 7148). Hence, in thisinvention, we are hereby disclaiming the content thereof which isincorporated by reference.

The following examples are offered by way of illustration, not bylimitation.

In the following list, selected PEGylation reagents are listed asactivated N-hydroxysuccinimide esters (OSu). Obviously, other activeesters may be employed, such as 4-nitrophenoxy and many other activeesters known to those skilled in the art. The PEG (or mPEG) moiety,CH₃O—(CH₂CH₂O)_(n)—, can be of any size up to Mw 40.000 Da, e.g., 750Da, 2000 Da, 5000 Da, 20.000 Da and 40.000 Da. The mPEG moiety can bepolydisperse but also monodisperse consisting of mPEG's with welldefined chain lengths (and, thus, molecular weights) of, e.g., 12 or 24repeating ethylene glycol units—denoted mdPEGx for m: methyl/methoxyend-capped, d: discrete and x for the number of repeating ethyleneglucol residues, e.g., 12 or 24. The PEG moiety can be either straightchain or branched. The structure/sequence of the PEG-residue on theextended insulin can formally be obtained by replacing the leaving group(e.g., “—OSu”) from the various PEGylation reagents with “NH-insulin”,where the insulin is PEGylated either in an epsilon position in a lysineresidue or in the alpha-amino position in the A- or B-chain (or both):

mPEG-COCH₂CH₂CO—OSu,mPEG-COCH₂CH₂CH₂CO—OSu,mPEG-CH₂CO—OSu,mPEG-CH₂CH₂CO—OSu,mPEG-CH₂CH₂CH₂CO—OSu,mPEG-CH₂CH₂CH₂CH₂CO—OSu,mPEG-CH₂CH₂CH₂CH₂CH₂CO—OSu,mPEG-CH₂CH(CH₃)CO—OSu,mPEG-CH₂CH₂CH(CH₃)CO—OSu,mPEG-CH₂CH₂NH—COCH₂CH₂CO—OSu,mPEG-CH₂CH₂CH₂NH—COCH₂CH₂CH₂CO—OSu,mPEG-CH₂CH₂CH₂NH—COCH₂CH₂CO—OSu,mPEG-CH₂CH₂NH—COCH₂CH₂CH₂CO—OSu,mPEG-CO-(4-nitrophenoxy),(mdPEG₁₂-CH₂CH₂NHCOCH₂CH₂OCH₂)₃CNHCOCH₂CH₂(OCH₂CH₂)₄NHCOCH₂CH₂CO—OSu(or, in short: (mdPEG₁₂)-3-dPEG₄-OSu),(mdPEG₁₂-CH₂CH₂NHCOCH₂CH₂OCH₂)₃CNHCOCH₂CH₂(OCH₂CH₂)₄NHCOCH₂CH₂CH₂CO—OSu(or, in short: (mdPEG₁₂)-3-dPEG₄-OSu),mdPEGx-COCH₂CH₂CO—OSu,mdPEGx-COCH₂CH₂CH₂CO—OSu,mdPEGx-CH₂CO—OSu,mdPEGx-CH₂CH₂CO—OSu,mdPEGx-CH₂CH₂CH₂CO—OSu,mdPEGx-CH₂CH₂CH₂CH₂CO—OSu,mdPEGx-CH₂CH₂CH₂CH₂CH₂CO—OSu,mdPEGx-CH₂CH(CH₃)CO—OSu,mdPEGx-CH₂CH₂CH(CH₃)CO—OSu,mdPEGx-CH₂CH₂NH—COCH₂CH₂CO—OSu,mdPEGx-CH₂CH₂CH₂NH—COCH₂CH₂CH₂CO—OSu,mdPEGx-CH₂CH₂CH₂NH—COCH₂CH₂CO—OSu,mdPEGx-CH₂CH₂NH—COCH₂CH₂CH₂CO—OSu,mdPEGx-CO-(4-nitrophenoxy),wherein x is an integer in the range from about 6 to about 48, e.g., 12or 24.

In addition, larger PEGylation reagents can be prepared by assemblingtwo or more smaller PEG reagents. For example, end-capped PEG reagentsas N-hydroxysuccinimide esters like any of the ones above can be coupledto—optionally protected—PEG moieties that are functionalised byamino-groups in one end and carboxylic acid (esters) in the other end.After deprotection of the carboxylic acid (if necessary) the carboxylicacid is activated eg. as the N-hydroxysuccinimide ester to furnish alonger PEGylation reagent. If desired, the obtained PEGylation reagentcan be further extended by repeating the cycle one or more times. Thisprinciple and methodology is illustrated in the examples.

This methodology enables construction of larger monodisperse (andpolydisperse) PEGylation reagents of tailored sizes.

Examples of PEG residues of the invention includes:

mPEG750 (where “750” indicates the average molecular weight in Da),mPEG2000,mPEG5000,mPEG10000,mPEG20000,mPEG30000,mPEG40000,mdPEG₁₂, (wherein “12” in subscript indicates the number of PEGmonomers—as defined herein and eg. by Quanta BioDesign Ltd.)mdPEG₂₄,mdPEG_(3x12) (wherein “3×12” in subscript indicates that PEG is branchedand composed of 3 arms each composed of 12 PEG monomers—as definedherein and eg. by Quanta BioDesign Ltd.),mdPEG₁₂-dPEG₁₂,mdPEG₁₂-dPEG₂₄,mdPEG₂₄-dPEG₁₂,mdPEG₂₄-dPEG₂₄,mdPEG₂₄-dPEG₂₄-dPEG₂₄,mdPEG_(3x12)-dPEG₁₂,mdPEG_(3x12)-dPEG₂₄-dPEG₂₄

In the following, selected PEGylation reagents are listed as maleimidederivatives. Obviously, as alternatives to the maleimide group, otherMichael acceptors may be employed, such as vinylsulfones and many otherMichael acceptors known to those skilled in the art. The PEG (or mPEG)moiety, CH₃O—(CH₂CH₂O)_(n)—, can be of any size up to Mw about 40.000Da. The structure/sequence of the PEG-residue on the extended insulincan formally be obtained by replacing the maleimide “MAL” from thevarious PEGylation reagents with “3-thio-succinimidyl-Ala-insulin”,where the insulin is PEGylated at a free cysteine residue according tothe scheme below:

This scheme illustrates PEGylation on a terminal Cys. Obviously, Cysneed not be placed terminally to enable PEGylation.

Example of PEG-MAL: mPEG-MAL.

The PEGylated, extended insulins of this invention have in the followingall been named as if the linker connecting the PEG moiety to the insulinin all cases is a (3-)propionyl linker (—CH₂—CH₂—CO—). It is evidentfrom the foregoing that many types of linkers are commercially availableand since it is not the exact structure/composition of the linker thatgoverns the beneficial effects of placing the PEG moiety at residuesoutside the sequence of regular insulin, it is to be understood that alltypes of linkers (cf. above) are within the scope of this invention.

Parent extended insulins of the invention comprise the following:

A22K, B29R, desB30 human insulin;A21Q, A22G, A23K, B29R, desB30 human insulin;A21G, A22G, A23K, B29R, desB30 human insulin;A22G, A23K, B29R, desB30 human insulin;A21Q, A22G, A23G, A24K, B29R, desB30 human insulin;A21G, A22G, A23G, A24K, B29R, desB30 human insulin;A21Q, A22G, A23G, A24G, A25K, B29R, desB30 human insulin;A21G, A22G, A23G, A24G, A25K, B29R, desB30 human insulin;A21G, A22K, B29R, desB30 human insulin;A21G, A22G, A23K, B29R, desB30 human insulin;A21G, A22G, A23G, A24K, B29R, desB30 human insulin;A21G, A22G, A23G, A24G, A25K, B29R, desB30 human insulin;A21Q, A22K, B29R, desB30 human insulin;A21Q, A22G, A23K, B29R, desB30 human insulin;A21Q, A22G, A23G, A24K, B29R, desB30 human insulin;A21Q, A22G, A23G, A24G, A25K, B29R, desB30 human insulin;A14E, A22K, B25H, B29R, desB30 human insulin;A14E, A21Q, A22K, B25H, B29R, desB30 human insulin;A14E, A21G, A22K, B25H, B29R, desB30 human insulin;A14E, A21Q, A22G, A23K, B25H, B29R, desB30 human insulin;A14E, A21G, A22G, A23K, B25H, B29R, desB30 human insulin;A14E, A21Q, A22G, A23G, A24K, B25H, B29R, desB30 human insulin;A14E, A21G, A22G, A23G, A24K, B25H, B29R, desB30 human insulin;A14E, A21Q, A22G, A23G, A24G, A25K, B25H, B29R, desB30 human insulin;A14E, A21G, A22G, A23G, A24G, A25K, B25H, B29R, desB30 human insulin;B29Q, B31K human insulin;A22K, B3Q, B29R, desB30 human insulin;A22K, B3S, B29R, desB30 human insulin;A22K, B3T, B29R, desB30 human insulin;A22K, B1Q, B29R, desB30 human insulin;A18Q, A22K, B29R, desB30 human insulin;A22K, desB1, B3Q, B29R, desB30 human insulin;A21G, B29Q, B31K human insulin;A21A, B29Q, B31K human insulin;A21Q, B29Q, B31K human insulin;A-1K, desB30 human insulin;A-1K, B29R, desB30 human insulin;A-3G, A-2G, A-1R desB30 human insulin; (for N-terminal A-3-PEGylation)A22K, B28E, B29R, desB30 human insulin;A22K, B28D, B29R, desB30 human insulin;A22K, desB27, B28E, B29R, desB30 human insulin;B28E, B29Q, B31K human insulin;desB27, B28E, B29Q, B31K human insulin;A22K, B28R, desB29, desB30 human insulin;B28R, B29P, B31K human insulin;A22K, B3Q, B28E, B29R, desB30 human insulin;A21G, B3Q, B28E, B29Q, B31K human insulin;A22K, B13Q, B29R, desB30 human insulin;A22K, desB1, B29R, desB30 human insulin;A14E, A22K, B25H, desB30 human insulin;A14E, B25H, B29Q, B31K human insulin;A13E, A22K, B25H, desB30 human insulin;A21Q, A22G, A23K, B29R, desB30 human insulin;A21Q, A22G, A23G, A24K, B29R, desB30 human insulin;A21Q, A22G, A23G, A24G, A25K, B29R, desB30 human insulin;A21A, A22K, B29R, desB30 human insulin;A21A, A22G, A23K, B29R, desB30 human insulin;A21G, A22G, A23K, B29R, desB30 human insulin;A21A, A22G, A23G, A24K, B29R, desB30 human insulin;A21G, A22G, A23G, A24K, B29R, desB30 human insulin;A21G, A22G, A23G, A24G, A25K, B29R, desB30 human insulin;A21A, A22G, A23G, A24G, A25K, B29R, desB30 human insulin;A21Q, A22K, B3Q, B29R, desB30 human insulin;A21A, A22K, B3Q, B29R, desB30 human insulin;A21G, A22K, B3Q, B29R, desB30 human insulin.

EXAMPLES General Procedures Construction of Expression Vectors,Transformation of the Yeast Cells, and Expression of the InsulinPrecursors of the Invention

All expressions plasmids are of the C-POT type, similar to thosedescribed in EP 171142, which are characterized by containing theSchizosaccharomyces pombe triose phosphate isomerase gene (POT) for thepurpose of plasmid selection and stabilization in S. cerevisiae. Theplasmids also contain the S. cerevisiae triose phosphate isomerasepromoter and terminator. These sequences are similar to thecorresponding sequences in plasmid pKFN1003 (described in WO 90/10075)as are all sequences except the sequence of the EcoRI-XbaI fragmentencoding the fusion protein of the leader and the insulin product. Inorder to express different fusion proteins, the EcoRI-XbaI fragment ofpKFN1003 is simply replaced by an EcoRI-XbaI fragment encoding theleader-insulin fusion of interest. Such EcoRI-XbaI fragments may besynthesized using synthetic oligonucleotides and PCR according tostandard techniques.

Yeast transformants were prepared by transformation of the host strainS. cerevisiae strain MT663 (MATa/MATα pep4-3/pep4-3 HIS4/his4tpi::LEU2/tpi::LEU2 Cir⁺). The yeast strain MT663 was deposited in theDeutsche Sammlung von Mikroorganismen und Zellkulturen in connectionwith filing WO 92/11378 and was given the deposit number DSM 6278.

MT663 was grown on YPGaL (1% Bacto yeast extract, 2% Bacto peptone, 2%galactose, 1% lactate) to an O.D. at 600 nm of 0.6. 100 ml of culturewas harvested by centrifugation, washed with 10 ml of water,recentrifuged and resuspended in 10 ml of a solution containing 1.2 Msorbitol, 25 mM Na₂EDTA pH=8.0 and 6.7 mg/ml dithiotreitol. Thesuspension was incubated at 30° C. for 15 minutes, centrifuged and thecells resuspended in 10 ml of a solution containing 1.2 M sorbitol, 10mM Na₂EDTA, 0.1 M sodium citrate, pH 05.8, and 2 mg Novozym®234. Thesuspension was incubated at 30° C. for 30 minutes, the cells collectedby centrifugation, washed in 10 ml of 1.2 M sorbitol and 10 ml of CAS(1.2 M sorbitol, 10 mM CaCl₂, 10 mM Tris HCl (pH=7.5) and resuspended in2 ml of CAS. For transformation, 1 ml of CAS-suspended cells was mixedwith approx. 0.1 mg of plasmid DNA and left at room temperature for 15minutes. 1 ml of (20% polyethylene glycol 4000, 10 mM CaCl₂, 10 mM TrisHCl, pH=7.5) was added and the mixture left for a further 30 minutes atroom temperature. The mixture was centrifuged and the pellet resuspendedin 0.1 ml of SOS (1.2 M sorbitol, 33% v/v YPD, 6.7 mM CaCl₂) andincubated at 30° C. for 2 hours. The suspension was then centrifuged andthe pellet resuspended in 0.5 ml of 1.2 M sorbitol. Then, 6 ml of topagar (the SC medium of Sherman et al. (1982) Methods in Yeast Genetics,Cold Spring Harbor Laboratory) containing 1.2 M sorbitol plus 2.5% agar)at 52° C. was added and the suspension poured on top of platescontaining the same agarsolidified, sorbitol containing medium. S.cerevisiae strain MT663 transformed with expression plasmids was grownin YPD for 72 h at 30° C.

Production, Purification and Characterization of the PEGylated InsulinDerivatives of the Invention

A number of insulin precursors were produced as described above andisolated from the culture medium and purified. The insulin precursorswere PEGylated and processed as described in the examples below toproduce the final insulin derivatives (General Procedure (A)).Optionally, the precursors can be processed by trypsin prior toPEGylation (General Procedure (B)). These insulin derivatives weretested for biological insulin activity as measured by binding affinityto the human insulin receptor relative to that of human insulin asdescribed below.

The following examples refer to intermediate compounds and finalproducts identified in the specification and in the examples. Thepreparation of the insulin derivatives of this invention is described indetail using the following examples, but the chemical reactions andpurification schemes described are disclosed in terms of their generalapplicability to the preparation of the insulin derivatives of theinvention. Occasionally, the reaction may not be applicable as describedto each compound included within the disclosed scope of the invention.The compounds for which this occurs will be readily recognised by thoseskilled in the art. In these cases the reactions can be successfullyperformed by conventional modifications known to those skilled in theart, that is, by appropriate protection of interfering groups, bychanging to other conventional reagents, or by routine modification ofreaction conditions. Alternatively, other reactions disclosed herein orotherwise conventional will be applicable to the preparation of thecorresponding compounds of the invention. In all preparative methods,all starting materials are known or may easily be prepared from knownstarting materials. All temperatures are set forth in degrees Celsiusand unless otherwise indicated, all parts and percentages are by weightwhen referring to yields and all parts are by volume when referring tosolvents and eluents.

The insulin derivatives of this invention can be purified by employingone or more of the following procedures which are typical within theart. These procedures can—if needed—be modified with regard togradients, pH, salts, concentrations, flow, columns and so forth.Depending on factors such as impurity profile, solubility of theinsulins in question etcetera, these modifications can readily berecognised and made by a person skilled in the art.

General Procedure (A) for Preparation of PEGylated, Extended Insulins ofthis Invention

The general procedure (A) is outlined below and illustrated in the firstexample:

Example 1 General Procedure (A)

A22K(N^(ε)-mPEG2000-Propionyl), B29R, desB30 Human Insulin

Step 1: Preparation and Purification of the Insulin Precursor LysA22ArqB29 B29R desB30 B′A

The insulin precursor A22K, B29R, desB30, B′A single chain insulin canbe purified as described in the purification steps A to C below.

Purification step A: Capture

In step A, 10.75 litres of cleared culture media is diluted by additionof 4.5 litres of 99% ethanol, to give a total volume of 15.25 litrescontaining 30 vol % ethanol (conductivity 2.7 mS/cm, pH=3.4). A 300 mlSP Big Beads Sepharose column (100-300 μm, Amersham Biosciences) wasequilibrated with 1 litre of 0.1 M citric acid pH 3.5 (flow app. 20ml/min), before loading the 15.25 litres of prepared culture media overnight (flow app. 10 ml/min). After loading the column was again washedwith 1 litre of 0.1 M citric acid pH 3.5 followed by 1 liter of 40 vol %ethanol (flow app. 20 ml/min). The bound insulin precursor A22K, B29R,desB30, B′A single chain insulin was then eluted with 1.5 litres of 0.2M sodium acetate, 35 vol % ethanol, pH 5.75 (flow: 1.5 ml/min, volume ofeluted precursor: 400 ml, amount of precursor: 220 mg).

Purification Step B: Reverse-Phase HPLC

In step B the eluate was evaporated to dryness and the pelletre-dissolved in 0.25 M acetic acid. The pH was lowered further to 1.5immediately before purification by reverse-phase HPLC on a C18 column(ODDMS C18, 20×250 mm, 200 Å, 10 μm, FeF Chemicals A/S). Beforeapplication to the column the precursor solution was sterile filtrated(22 μm, Low Protein Binding Durapore® (PVDF), Millipore). A gradientfrom 15% B to 50% B was run over the column, where Buffer A: 0.2 M(NH₄)₂SO₄, 0.04 M ortho-phosphoric acid, 10 vol % ethanol, pH 2.5 andBuffer B: 70 vol % ethanol. The gradient was run over 120 min with aflow of 5 ml/min, column temperature at 40° C. The insulin precursorA22K, B29R, desB30, B′A single chain insulin was eluted and pooled(total volume 75 ml).

Purification Step C: De-Salting by Gelfiltration

In step C the ethanol content in the eluate from reverse-phase HPLC waslowered to less than 5 vol % using a rotary evaporator (new volume: ˜50ml). A 1000 ml G25 Sephadex column (5×55 cm, Amersham Biosciences) waswashed in 0.5 M acetic acid and the insulin precursor A22K, B29R,desB30, B′A single chain insulin was then applied to the column andthereby de-salted by gelfiltration in 0.5 M acetic acid. The insulinprecursor was followed by UV detection at 280 nm, while the salt wasfollowed by conductivity measurement. Immediately after de-salting, theinsulin precursor was lyophilized.

Step 2: Synthesis of A22K(N^(ε)-mPEG2000-propionyl), B29R, desB30 B′Ahuman insulin precursor 0.15 mmol of lyophilized insulin precursorLysA22 ArgB29 desB30 B′A is dissolved in aqueous sodium carbonate (3 ml,100 mM). A solution of the PEGylation reagent mPEG2000-SPA-OSu inacetonitrile (0.15 mmol in 3 ml) is added to the solution of theprecursor, and the mixture is gently stirred for 1 hour. The mixture islyophilised, purified by HPLC and lyophilised to afford the PEGylatedprecursor.Step 3: Conversion to A22K(N^(ε)-mPEG2000-Propionyl), B29R, desB30 HumanInsulin

The PEGylated insulin precursor A22K(N^(ε)-mPEG2000-propionyl), desB30B′A single chain human insulin precursor (3.9 μmol) is dissolved in 4.2ml 50 mM glycine, 20 vol % ethanol pH 10.0. 3.6 mg of lyophilizedporcine trypsin (Novo Nordisk A/S) is also dissolved in 3.5 ml 50 mMglycine, 20 vol % ethanol pH 10.0. Of this trypsin solution 0.5 ml isthen added to the insulin precursor solution (hereby the insulinprecursor is in 200 times excess). The mixture is then incubated at roomtemperature for 15 minutes, after which the trypsin activity is stoppedby lowering the pH<3 (pH=2.08 with 0.5 M acetic acid). The PEGylatedinsulin analogue A22K(N^(ε)-mPEG2000-propionyl), B29R, desB30 humaninsulin is then purified (removing trypsin and any un-acylated,doubly-acylated etc. or un-cleaved insulin molecules) by reverse-phaseHPLC an lyophilised to afford the title insulin.

General Procedure (B) for Preparation of PEGylated, Extended Insulins ofthis Invention

This procedure is quite similar to General Procedure (A). The order ofthe individual steps has been changed, so that the B′A-precursors arecleaved by trypsin prior to PEGylation. The general procedure (B) isillustrated in the first example.

Example 2 General Procedure (B)

A22K(N^(ε)mPEG2000-propionyl), B29R, desB30 Human Insulin

A22K, B29R, desB30 human insulin (125 mg) was dissolved in 0.1 M Na₂CO₃(2.8 ml). mPEG-SPA 2000 (50 mg) dissolved in acetonitrile (1.25 ml) wasadded. pH was adjusted from 10.2 to 10.4 with 0.1 N NaOH. After 50 minmore mPEG-SPA 2000 (25 mg) dissolved in acetonitrile (1.25 ml) wasadded. After slow stirring for 80 min, water (4.5 ml) was added and pHwas adjusted to 5 with 1 N HCl. The mixture was lyophilized. The titlecompound was obtained by preparative HPLC purification. Column: C4, 2cm. A-Buffer: 0.1% TFA in MiliQ Water; B-buffer: 0.1% TFA inacetonitrile. Gradient 30-65% B over 30 min. Yield 43 mg.

MALDI-MS (matrix: sinapinic acid); m/z: 8114.

Example 3 General Procedure (B)

A22K(N^(ε)mPEG750-Propionyl, B29R, desB30 Human Insulin

MALDI-MS (matrix: sinapinic acid); m/z: 5862.

Example 4 General Procedure (B)

A22K(N^(ε)mdPEG12-Propionyl, B29R, desB30 Human Insulin

MALDI (matrix: sinapinic acid); m/z: 6432.

Example 5 General Procedure (B)

A22K(N^(ε)mdPEG₂₄-Propionyl, B29R, desB30 Human Insulin

MALDI (matrix: sinapinic acid); m/z: 6962.

Example 6 General Procedure (B)

A22K(N^(ε)(mdPEG₁₂)₃-dPEG₄-yl), B29R, desB30 Human Insulin

MALDI-MS (matrix: sinapinic acid); m/z: 8167.

Example 7 General Procedure (B)

A22G, A23G, A24G, A25K(N^(ε)mPEG750-Propionyl), B29R, desB30 HumanInsulin

MALDI-MS (matrix: sinapinic acid); m/z: 6807

Example 8 General Procedure (B)

A22G, A23K(N^(ε)mPEG2000-Propionyl), B29R, desB30 human insulin

MALDI-MS (matrix: sinapinic acid); m/z: 8170

Example 9 General Procedure (B)

A22K(N^(ε)mdPEG₈-Propionyl), B29R, desB30 Human Insulin

MALDI-MS (matrix: sinapinic acid); m/z: 6258.

Example 10 General Procedure (B)

A22K(N^(ε)mdPEG₄-Propionyl), B29R, desB30 Human Insulin

MALDI-MS (matrix: sinapinic acid); m/z: 6082.

Example 11 General Procedure (B)

A22K(N^(ε)mPEG5000-Propionyl), B29R, desB30 human insulin

MALDI-MS (matrix: sinapinic acid); m/z: around 11600.

Example 12 General Procedure (B)

A22K(N^(ε)mPEG20000-Propionyl), B29R, desB30 human insulin

MALDI-MS (matrix: sinapinic acid); m/z: around 21500.

Example 13 General Procedure (B)

A14E, A22K(N^(ε)mdPEG₁₂-Propionyl), B25H, B29R, desB30 Human Insulin

MALDI-MS (matrix: sinapinic acid); m/z: 7520.

Example 14 General Procedure (B)

A14E, A22K(N^(ε)mdPEG₁₂-dPEG₂₄-Propionyl), B25H, B29R, desB30 humaninsulin

MALDI-MS (matrix: sinapinic acid); m/z: 7520.

The PEGylation reagent was prepared as described in the following:

Preparation of omega-(methoxy-PEG₁₁-propanoylamino)-PEG₂₄-Propanoic Acid(mdPEG₁₂-dPEG₂₄ Acid)

mdPEG₁₂ NHS ester (0.457 mmol, Quanta BioDesign Ltd. Product No 10262)and amino-dPEG₂₄ tert-butylester (0.416 mmol, Quanta BioDesign, ProductNo 10311) were dissolved separately in acetonitrile (each 10 mL) andthen the two solutions were mixed, pH was adjusted with DIPEA to pH 8(measurement of pH was done using wet indicator strips). The resultingmixture was stirred at RT overnight, and subsequently evaporated todryness, followed by treatment with TFA/DCM (1/1), 10 mL for 1 h at RT.The mixture was then evaporated to dryness and stripped twice with DCM.The residue was purified by HPLC (2 cm, C18 column) using acetonitrile(AcCN)/0, 1% TFA and water/0, 1% TFA as eluents. Gradient: 10-80%AcCN/TFA from 5-20 min. Fractions containing the desired compound werecollected, combined and evaporated to dryness resulting inomega-(methoxy-PEGL-Dropanoylamino)PEG₂₃-propanoic acid as an oil (249mg, 35%).

LCMS: m/z: 1718 (M+1)⁺.

Preparation of omega-(methoxy-PEG₁₁-propanoylamino)-PEG₂₄-Propanoic AcidN-hydroxysuccinimide Ester (mdPEG₁₂-dPEG₂₄-NHS ormdPEG₁₂-dPEG₂₄-propanoic Acid OSu Ester)

Omega-(methoxy-PEG₁₁-propanoylamino)PEG₂₄-propanoic acid (249 mg, 0.145mmol) was dissolved in acetonitrile (10 mL) and pH was adjusted to 8 byaddition of DIPEA (measurement of pH was done using wet indicatorstrips). TSTU (48 mg, 0.16 mmol) in acetonitrile (10 mL) was added andthe mixture was stirred at room temperature for 1.5 h, and evaporated todryness. The residue was dissolved in DCM and washed with hydrochloricacid (0.01 M), the organic phase was dried (MgSO₄), filtered and thefiltrate was evaporated to dryness. The resultingomega-(methoxy-PEG₁₁-propanoylamino)PEG₂₄ propanoic acidN-hydroxysuccinimide ester was used for coupling to insulin withoutfurther purification.

LCMS: m/z 1813.8 (M+1)⁺.

Example 15 General Procedure (B)

A14E, A22K(N^(ε)mdPEG₂₄-dPEG₁₂-Propionyl), B25H, B29R, desB30 HumanInsulin

MALDI-MS (matrix: sinapinic acid); m/z: 7519.

The N-hydroxysuccinimide activated PEG reagent was prepared similarly asdescribed above from mdPEG₂₄ NHS ester (Quanta BiodDesign Ltd. ProductNo 10304) and amino-dPEG₁₂ tert-butyl ester (Quanta BioDesign Ltd.Product No 10281) via omega-(methoxy-PEG₂₃-propanoylamino)PEG₁₂propanoic acid tert-butyl ester,omega-(methoxy-PEG₂₃-propanoylamino)PEG₁₂ propanoic acid, andomega-(methoxy-PEG₂₃-propanoylamino)PEG₁₂ propanoic acid NHS ester

LCMS: m/z 1814 (M+1)⁺.

Example 16 General Procedure (B)

A14E, A22K(N^(ε)mPEG2000-Propionyl), B25H, B29R, desB30 Human Insulin

MALDI-MS (matrix: sinapinic acid); m/z: around 8200.

Example 17 General Procedure (B)

A14E, A22K(N^(ε)(mdPEG₁₂)₃-dPEG₄-yl), B25H, B29R, desB30 Human Insulin

MALDI-MS (matrix: sinapinic acid); m/z: 8123.

This insulin was prepared using the PEG reagent NHS-dPEG₄-(m-dPEG₁₂)₃ester (Quanta BioDesign Ltd. Product No 10401).

Example 18 General Procedure (B)

A14E, A22K(N^(ε)(mdPEG₁₂)₃-dPEG₄-yl-dPEG12-yl), B25H, B29R, desB30 Humaninsulin

MALDI-MS (matrix: sinapinic acid); m/z: 8724.

This insulin was prepared using the PEG reagent NHS-dPEG₄-(m-dPEG₁₂)₃ester (Quanta BioDesign Ltd. Product No 10401) and amino-dPEG₁₂tert-butyl ester (Quanta BioDesign Product No 10281) similarly asdescribed above.

Example 19 General Procedure (B)

A22K(N^(ε)(mdPEG₁₂)₃-dPEG₄-yl-dPEG12-yl), B29R, desB30 Human Insulin

MALDI-MS (matrix: sinapinic acid); m/z: 8768.

This insulin was prepared using the PEG reagent NHS-dPEG₄-(m-dPEG₁₂)₃ester (Quanta BioDesign Ltd. Product No 10401) and amino-dPEG₁₂tert-butyl ester (Quanta BioDesign Product No 10281) similarly asdescribed above.

Example 20 General Procedure (B)

A22K(N^(ε)(mdPEG₂₄-Propionyl), A14E, B25H, B29R, desB30 human insulin

MALDI-MS (matrix: sinapinic acid); m/z: 6918.

Example 21 General Procedure (B)

A22K(N^(ε)(mPEG5.000-Propionyl)), A14E, B25H, B29R, desB30 human insulin

MALDI-MS (matrix: sinapinic acid); m/z: around 11400.

Example 22 General Procedure (B)

B29Q, B31K(N/(mPEG2000-Propionyl)) human insulin

MALDI-MS (matrix: sinapinic acid); m/z: around 8268.

Example 23 Insulin Receptor Binding of the Insulin Derivatives of thisInvention

The affinity of the insulin derivatives of this invention for the humaninsulin receptor is determined by a SPA assay (Scintillation ProximityAssay) microtiterplate antibody capture assay. SPA-PVT antibody-bindingbeads, anti-mouse reagent (Amersham Biosciences, Cat No. PRNQ0017) aremixed with 25 ml of binding buffer (100 mM HEPES pH 7.8; 100 mM sodiumchloride, 10 mM MgSO₄, 0.025% Tween-20). Reagent mix for a singlePackard Optiplate (Packard No. 6005190) is composed of 2.4 μl of a1:5000 diluted purified recombinant human insulin receptor (either withor without exon 11), an amount of a stock solution of A14Tyr[¹²⁵]-humaninsulin corresponding to 5000 cpm per 100 μl of reagent mix, 12 μl of a1:1000 dilution of F12 antibody, 3 ml of SPA-beads and binding buffer toa total of 12 ml. A total of 100 μl reagent mix is then added to eachwell in the Packard Optiplate and a dilution series of the insulinderivative is made in the Optiplate from appropriate samples. Thesamples are then incubated for 16 hours while gently shaken. The phasesare the then separated by centrifugation for 1 min and the platescounted in a Topcounter. The binding data were fitted using thenonlinear regression algorithm in the GraphPad Prism 2.01 (GraphPadSoftware, San Diego, Calif.).

Insulin Receptor Binding Affinities of Selected Compounds of thisInvention:

Insulin receptor binding, A-isoform (without exon 11) Ex. No: Relativeto human insulin: 1, 2 90%  3 123%  4 188%  6 120%  5 118%  7 44%  8 58% 9 128% 10 123% 15 20% 13 24% 14 16% 17 19% 18 16% 19 106% 20 24% 22 14%16 15%

Example 24 Blood Glucose Lowering Effect After i.v. Bolus Injection inRat of the Insulin Derivatives of this Invention

Male Wistar rats, 200-300 g, fasted for 18 h, is anesthetized usingeither Hypnorm-Dormicum s.c. (1.25 mg/ml Dormicum, 2.5 mg/ml fluanisone,0.079 mg/ml fentanyl citrate) 2 ml/kg as a priming dose (to timepoint−30 min prior to test substance dosing) and additional 1 ml/kg every 20minutes.

The animals are dosed with an intravenous injection (tail vein), 1ml/kg, of control and test compounds (usual dose range 0.125-20nmol/kg). Blood samples for the determination of whole blood glucoseconcentration are collected in heparinized 10 μl glass tubes by punctureof the capillary vessels in the tail tip to time −20 min and 0 min(before dosing), and to time 10, 20, 30, 40, 60, 80, 120, and 180 minafter dosing. Blood glucose concentrations are measured after dilutionin analysis buffer by the immobilized glucose oxidase method using anEBIO Plus autoanalyzer (Eppendorf, Germany). Mean plasma glucoseconcentrations courses (mean±SEM) are made for each dose and eachcompound.

Example 25 Potency of the Insulin Derivatives of this Invention Relativeto Human Insulin

Sprague Dawley male rats weighing 238-383 g on the experimental day areused for the clamp experiment. The rats have free access to feed undercontrolled ambient conditions and are fasted overnight (from 3 μm) priorto the clamp experiment.

Experimental Protocol:

The rats are acclimatized in the animal facilities for at least 1 weekprior to the surgical procedure. Approximately 1 week prior to the clampexperiment, Tygon catheters are inserted under halothane anaesthesiainto the jugular vein (for infusion) and the carotid artery (for bloodsampling) and exteriorised and fixed on the back of the neck. The ratsare given Streptocilin vet. (Boehringer Ingelheim; 0.15 ml/rat, i.m.)post-surgically and placed in an animal care unit (25° C.) during therecovery period. In order to obtain analgesia, Anorphin (0.06 mg/rat,s.c.) is administered during anaesthesia and Rimadyl (1.5 mg/kg, s.c.)is administered after full recovery from the anaesthesia (2-3 h) andagain once daily for 2 days.

At 7 am on the experimental day overnight fasted (from 3 μm the previousday) rats are weighed and connected to the sampling syringes andinfusion system (Harvard 22 Basic pumps, Harvard, and PerfectumHypodermic glass syringe, Aldrich) and then placed into individual clampcages where they rest for ca. 45 min before start of experiment. Therats are able to move freely on their usual bedding during the entireexperiment and have free access to drinking water. After a 30 min basalperiod during which plasma glucose levels were measured at 10 minintervals, the insulin derivative to be tested and human insulin (onedose level per rat, n=6-7 per dose level) are infused (i.v.) at aconstant rate for 300 min. Plasma glucose levels are measured at 10 minintervals throughout and infusion of 20% aqueous glucose is adjustedaccordingly in order to maintain euglyceamia. Samples of re-suspendederythrocytes are pooled from each rat and returned in about ½ ml volumesvia the α-rotid catheter.

On each experimental day, samples of the solutions of the individualinsulin derivatives to be tested and the human insulin solution aretaken before and at the end of the clamp experiments and theconcentrations of the peptides are confirmed by HPLC. Plasmaconcentrations of rat insulin and C-peptide as well as of the insulinderivative to be tested and human insulin are measured at relevant timepoints before and at the end of the studies. Rats are killed at the endof experiment using a pentobarbital overdose.

Example 26 Pulmonary Delivery of Insulin Derivatives to Rats

The test substance will be dosed pulmonary by the drop instillationmethod. In brief, male Wistar rats (app.250 g) are anaesthesized in app.60 ml fentanyl/dehydrodenzperidol/-dormicum given as a 6.6 ml/kg scprimingdose and followed by 3 maintenance doses of 3.3 ml/kg sc with aninterval of 30 min. Ten minutes after the induction of anaesthesia,basal samples are obtained from the tail vein (t=−20 min) followed by abasal sample immediately prior to the dosing of test substance (t=0). Att=0, the test substance is dosed intra tracheally into one lung. Aspecial cannula with rounded ending is mounted on a syringe containingthe 200 ul air and test substance (1 ml/kg). Via the orifice, thecannula is introduced into the trachea and is forwarded into one of themain bronchi—just passing the bifurcature. During the insertion, theneck is palpated from the exterior to assure intratracheal positioning.The content of the syringe is injected followed by 2 sec pause.Thereafter, the cannula is slowly drawn back. The rats are keptanaesthesized during the test (blood samples for up to 4 or 8 hrs) andare euthanized after the experiment.

The PEGylated extended insulins in the following examples may beprepared similarly as described above:

Examples 27-419

Ex. #: PEGylated, extended insulin: 27 A22K(N^(ε)mPEG10.000-propionyl)B29R desB30 human insulin; 28 A22K(N^(ε)mPEG40.000-propionyl) B29RdesB30 human insulin; 29 A22G A23K(N^(ε)mPEG750-propionyl) B29R desB30human insulin; 30 A22G A23K(N^(ε)mPEG5.000-propionyl) B29R desB30 humaninsulin; 31 A22G A23K(N^(ε)mPEG10.000-propionyl) B29R desB30 humaninsulin; 32 A22G A23K(N^(ε)mPEG20.000-propionyl) B29R desB30 humaninsulin; 33 A22G A23K(N^(ε)mPEG40.000-propionyl) B29R desB30 humaninsulin; 34 A22G A23K(N^(ε)mdPEG₁₂-propionyl) B29R desB30 human insulin;35 A22G A23K(N^(ε)mdPEG₂₄-propionyl) B29R desB30 human insulin; 36 A22GA23K((mdPEG₁₂)₃-dPEG₄-yl) B29R desB30 human insulin; 37 A22G A23GA24K(N^(ε)mPEG750-propionyl) B29R desB30 human insulin; 38 A22G A23GA24K(N^(ε)mPEG2.000-propionyl) B29R desB30 human insulin; 39 A22G A23GA24K(N^(ε)mPEG5.000-propionyl) B29R desB30 human insulin; 40 A22G A23GA24K(N^(ε)mPEG10.000-propionyl) B29R desB30 human insulin; 41 A22G A23GA24K(N^(ε)mPEG20.000-propionyl) B29R desB30 human insulin; 42 A22G A23GA24K(N^(ε)mPEG40.000-propionyl) B29R desB30 human insulin; 43 A22G A23GA24K(N^(ε)mdPEG₁₂-propionyl) B29R desB30 human insulin; 44 A22G A23GA24K(N^(ε)mdPEG₂₄-propionyl) B29R desB30 human insulin; 45 A22G A23GA24K(N^(ε)(mdPEG12)₃-dPEG₄-yl) B29R desB30 human insulin; 46 A22G A23GA24G A25K(N^(ε)mPEG2.000-propionyl) B29R desB30 human insulin; 47 A22GA23G A24G A25K(N^(ε)mPEG5.000-propionyl) B29R desB30 human insulin; 48A22G A23G A24G A25K(N^(ε)mPEG10.000-propionyl) B29R desB30 humaninsulin; 49 A22G A23G A24G A25K(N^(ε)mPEG20.000-propionyl) B29R desB30human insulin; 50 A22G A23G A24G A25K(N^(ε)mPEG40.000-propionyl) B29RdesB30 human insulin; 51 A22G A23G A24G A25K(N^(ε)mdPEG₁₂-propionyl)B29R desB30 human insulin; 52 A22G A23G A24GA25K(N^(ε)mdPEG₂₄-propionyl) B29R desB30 human insulin; 53 A22G A23GA24G A25K(N^(ε)(mdPEG₁₂)₃-dPEG₄-yl) B29R desB30 human insulin; 54A22K(N^(ε)mPEG750-propionyl) B3Q B29R desB30 human insulin; 55A22K(N^(ε)mPEG2.000-propionyl) B3Q B29R desB30 human insulin; 56A22K(N^(ε)mPEG5.000-propionyl) B3Q B29R desB30 human insulin; 57A22K(N^(ε)mPEG10.000-propionyl) B3Q B29R desB30 human insulin; 58A22K(N^(ε)mPEG20.000-propionyl) B3Q B29R desB30 human insulin; 59A22K(N^(ε)mPEG40.000-propionyl) B3Q B29R desB30 human insulin; 60A22K(N^(ε)mdPEG₁₂-propionyl) B3Q B29R desB30 human insulin; 61A22K(N^(ε)mdPEG₂₄-propionyl) B3Q B29R desB30 human insulin; 62A22K(N^(ε)(mdPEG₁₂)₃-dPEG₄-yl) B3Q B29R desB30 human insulin; 63A22K(N^(ε)mPEG750-propionyl) B3S B29R desB30 human insulin; 64A22K(N^(ε)mPEG2.000-propionyl) B3S B29R desB30 human insulin; 65A22K(N^(ε)mPEG5.000-propionyl) B3S B29R desB30 human insulin; 66A22K(N^(ε)mPEG10.000-propionyl) B3S B29R desB30 human insulin; 67A22K(N^(ε)mPEG20.000-propionyl) B3S B29R desB30 human insulin; 68A22K(N^(ε)mPEG40.000-propionyl) B3S B29R desB30 human insulin; 69A22K(N^(ε)mdPEG₁₂-propionyl) B3S B29R desB30 human insulin; 70A22K(N^(ε)mdPEG₂₄-propionyl) B3S B29R desB30 human insulin; 71A22K(N^(ε)(mdPEG₁₂)₃-dPEG₄-yl) B3S B29R desB30 human insulin; 72A22K(N^(ε)mPEG750-propionyl) B3T B29R desB30 human insulin; 73A22K(N^(ε)mPEG2.000-propionyl) B3T B29R desB30 human insulin; 74A22K(N^(ε)mPEG5.000-propionyl) B3T B29R desB30 human insulin; 75A22K(N^(ε)mPEG10.000-propionyl) B3T B29R desB30 human insulin; 76A22K(N^(ε)mPEG20.000-propionyl) B3T B29R desB30 human insulin; 77A22K(N^(ε)mPEG40.000-propionyl) B3T B29R desB30 human insulin; 78A22K(N^(ε)mdPEG₁₂-propionyl) B3T B29R desB30 human insulin; 79A22K(N^(ε)mdPEG₂₄-propionyl) B3T B29R desB30 human insulin; 80A22K(N^(ε)(mdPEG₁₂)₃-dPEG₄-yl) B3T B29R desB30 human insulin; 81A22K(N^(ε)mPEG750-propionyl) B1Q B29R desB30 human insulin; 82A22K(N^(ε)mPEG2.000-propionyl) B1Q B29R desB30 human insulin; 83A22K(N^(ε)mPEG5.000-propionyl) B1Q B29R desB30 human insulin; 84A22K(N^(ε)mPEG10.000-propionyl) B1Q B29R desB30 human insulin; 85A22K(N^(ε)mPEG20.000-propionyl) B1Q B29R desB30 human insulin; 86A22K(N^(ε)mPEG40.000-propionyl) B1Q B29R desB30 human insulin; 87A22K(N^(ε)mdPEG₁₂-propionyl) B1Q B29R desB30 human insulin; 88A22K(N^(ε)mdPEG₂₄-propionyl) B1Q B29R desB30 human insulin; 89A22K(N^(ε)(mdPEG₁₂)₃-dPEG₄-yl) B1Q B29R desB30 human insulin; 90 A18QA22K(N^(ε)mPEG750-propionyl) B29R desB30 human insulin; 91 A18QA22K(N^(ε)mPEG2.000-propionyl) B29R desB30 human insulin; 92 A18QA22K(N^(ε)mPEG5.000-propionyl) B29R desB30 human insulin; 93 A18QA22K(N^(ε)mPEG10.000-propionyl) B29R desB30 human insulin; 94 A18QA22K(N^(ε)mPEG20.000-propionyl) B29R desB30 human insulin; 95 A18QA22K(N^(ε)mPEG40.000-propionyl) B29R desB30 human insulin; 96 A18QA22K(N^(ε)mdPEG₁₂-propionyl) B29R desB30 human insulin; 97 A18QA22K(N^(ε)mdPEG₂₄-propionyl) B29R desB30 human insulin; 98 A18QA22K(N^(ε)(mdPEG₁₂)₃-dPEG₄-yl) B29R desB30 human insulin; 99A22K(N^(ε)mPEG750-propionyl) desB1 B3Q B29R desB30 human insulin; 100A22K(N^(ε)mPEG2.000-propionyl) desB1 B3Q B29R desB30 human insulin; 101A22K(N^(ε)mPEG5.000-propionyl) desB1 B3Q B29R desB30 human insulin; 102A22K(N^(ε)mPEG10.000-propionyl) desB1 B3Q B29R desB30 human insulin; 103A22K(N^(ε)mPEG20.000-propionyl) desB1 B3Q B29R desB30 human insulin; 104A22K(N^(ε)mPEG40.000-propionyl) desB1 B3Q B29R desB30 human insulin; 105A22K(N^(ε)mdPEG₁₂-propionyl) desB1 B3Q B29R desB30 human insulin; 106A22K(N^(ε)mdPEG₂₄-propionyl) desB1 B3Q B29R desB30 human insulin; 107A22K(N^(ε)(mdPEG₁₂)₃-dPEG₄-yl) desB1 B3Q B29R desB30 human insulin; 108B29Q B31K(N^(ε)mPEG750-propionyl) human insulin; 109 B29QB31K(N^(ε)mPEG2.000-propionyl) human insulin; 110 B29QB31K(N^(ε)mPEG5.000-propionyl) human insulin; 111 B29QB31K(N^(ε)mPEG10.000-propionyl) human insulin; 112 B29QB31K(N^(ε)mPEG20.000-propionyl) human insulin; 113 B29QB31K(N^(ε)mPEG40.000-propionyl) human insulin; 114 B29QB31K(N^(ε)mdPEG₁₂-propionyl) human insulin; 115 B29QB31K(N^(ε)mdPEG₂₄-propionyl) human insulin; 116 B29QB31K(N^(ε)(mdPEG₁₂)₃-dPEG₄-yl) human insulin; 117 A21G B29QB31K(N^(ε)mPEG750-propionyl) human insulin; 118 A21G B29QB31K(N^(ε)mPEG2.000-propionyl) human insulin; 119 A21G B29QB31K(N^(ε)mPEG5.000-propionyl) human insulin; 120 A21G B29QB31K(N^(ε)mPEG10.000-propionyl) human insulin; 121 A21G B29QB31K(N^(ε)mPEG20.000-propionyl) human insulin; 122 A21G B29QB31K(N^(ε)mPEG40.000-propionyl) human insulin; 123 A21G B29QB31K(N^(ε)mdPEG₁₂-propionyl) human insulin; 124 A21G B29QB31K(N^(ε)mdPEG₂₄-propionyl) human insulin; 125 A21G B29QB31K(N^(ε)(mdPEG₁₂)₃-dPEG₄-yl) human insulin; 126 A21A B29QB31K(N^(ε)mPEG750-propionyl) human insulin; 127 A21A B29QB31K(N^(ε)mPEG2.000-propionyl) human insulin; 128 A21A B29QB31K(N^(ε)mPEG5.000-propionyl) human insulin; 129 A21A B29QB31K(N^(ε)mPEG10.000-propionyl) human insulin; 130 A21A 29QB31K(N^(ε)mPEG20.000-propionyl) human insulin; 131 A21A B29QB31K(N^(ε)mPEG40.000-propionyl) human insulin; 132 A21A B29QB31K(N^(ε)mdPEG₁₂-propionyl) human insulin; 133 A21A B29QB31K(N^(ε)mdPEG₂₄-propionyl) human insulin; 134 A21A B29QB31K(N^(ε)(mdPEG₁₂)₃-dPEG₄-yl) human insulin; 135 A21Q B29QB31K(N^(ε)mPEG750-propionyl) human insulin; 136 A21Q B29QB31K(N^(ε)mPEG2.000-propionyl) human insulin; 137 A21Q B29QB31K(N^(ε)mPEG5.000-propionyl) human insulin; 138 A21Q B29QB31K(N^(ε)mPEG10.000-propionyl) human insulin; 139 A21Q B29QB31K(N^(ε)mPEG20.000-propionyl) human insulin; 140 A21Q B29QB31K(N^(ε)mPEG40.000-propionyl) human insulin; 141 A21Q B29QB31K(N^(ε)mdPEG₁₂-propionyl) human insulin; 142 A21Q B29QB31K(N^(ε)mdPEG₂₄-propionyl) human insulin; 143 A21Q B29QB31K(N^(ε)(mdPEG₁₂)₃-dPEG₄-yl) human insulin; 144A-1K(N^(ε)mPEG750-propionyl) desB30 human insulin; 145A-1K(N^(ε)mPEG2.000-propionyl) desB30 human insulin; 146A-1K(N^(ε)mPEG5.000-propionyl) desB30 human insulin; 147A-1K(N^(ε)mPEG10.000-propionyl) desB30 human insulin; 148A-1K(N^(ε)mPEG20.000-propionyl) desB30 human insulin; 149A-1K(N^(ε)mPEG40.000-propionyl) desB30 human insulin; 150A-1K(N^(ε)mdPEG₁₂-propionyl) desB30 human insulin; 151A-1K(N^(ε)mdPEG₂₄-propionyl) desB30 human insulin; 152A-1K(N^(ε)(mdPEG₁₂)₃-dPEG₄-yl) desB30 human insulin; 153A-1K(N^(ε)mPEG750-propionyl) B29R desB30 human insulin; 154A-1K(N^(ε)mPEG2.000-propionyl) B29R desB30 human insulin; 155A-1K(N^(ε)mPEG5.000-propionyl) B29R desB30 human insulin; 156A-1K(N^(ε)mPEG10.000-propionyl) B29R desB30 human insulin; 157A-1K(N^(ε)mPEG20.000-propionyl) B29R desB30 human insulin; 158A-1K(N^(ε)mPEG40.000-propionyl) B29R desB30 human insulin; 159A-1K(N^(ε)mdPEG₁₂-propionyl) B29R desB30 human insulin; 160A-1K(N^(ε)mdPEG₂₄-propionyl) B29R desB30 human insulin; 161A-1K(N^(ε)(mdPEG₁₂)₃-dPEG₄-yl) B29R desB30 human insulin; 162A-3G(N^(α)mPEG750-propionyl) A-2G A-1R desB30 human insulin; 163A-3G(N^(α)mPEG2.000-propionyl) A-2G A-1R desB30 human insulin; 164A-3G(N^(α)mPEG5.000-propionyl) A-2G A-1R desB30 human insulin; 165A-3G(N^(α)mPEG10.000-propionyl) A-2G A-1R desB30 human insulin; 166A-3G(N^(α)mPEG20.000-propionyl) A-2G A-1R desB30 human insulin; 167A-3G(N^(α)mPEG40.000-propionyl) A-2G A-1R desB30 human insulin; 168A-3G(N^(α)mdPEG₁₂-propionyl) A-2G A-1R desB30 human insulin; 169A-3G(N^(α)mdPEG₂₄-propionyl) A-2G A-1R desB30 human insulin; 170A-3G(N^(α)(mdPEG₁₂)₃-dPEG₄-yl) A-2G A-1R desB30 human insulin; 171A22K(N^(ε)mPEG750-propionyl) B28E B29R desB30 human insulin; 172A22K(N^(ε)mPEG2.000-propionyl) B28E B29R desB30 human insulin; 173A22K(N^(ε)mPEG5.000-propionyl) B28E B29R desB30 human insulin; 174A22K(N^(ε)mPEG10.000-propionyl) B28E B29R desB30 human insulin; 175A22K(N^(ε)mPEG20.000-propionyl) B28E B29R desB30 human insulin; 176A22K(N^(ε)mPEG40.000-propionyl) B28E B29R desB30 human insulin; 177A22K(N^(ε)mdPEG₁₂-propionyl) B28E B29R desB30 human insulin; 178A22K(N^(ε)mdPEG₂₄-propionyl) B28E B29R desB30 human insulin; 179A22K(N^(ε)(mdPEG₁₂)₃-dPEG₄-yl) B28E B29R desB30 human insulin; 180A22K(N^(ε)mPEG750-propionyl) B28D B29R desB30 human insulin; 181A22K(N^(ε)mPEG2.000-propionyl) B28D B29R desB30 human insulin; 182A22K(N^(ε)mPEG5.000-propionyl) B28D B29R desB30 human insulin; 183A22K(N^(ε)mPEG10.000-propionyl) B28D B29R desB30 human insulin; 184A22K(N^(ε)mPEG20.000-propionyl) B28D B29R desB30 human insulin; 185A22K(N^(ε)mPEG40.000-propionyl) B28D B29R desB30 human insulin; 186A22K(N^(ε)mdPEG₁₂-propionyl) B28D B29R desB30 human insulin; 187A22K(N^(ε)mdPEG₂₄-propionyl) B28D B29R desB30 human insulin; 188A22K(N^(ε)(mdPEG₁₂)₃-dPEG₄-yl) B28D B29R desB30 human insulin; 189A22K(N^(ε)mPEG750-propionyl) desB27 B28E B29R desB30 human insulin; 190A22K(N^(ε)mPEG2.000-propionyl) desB27 B28E B29R desB30 human insulin;191 A22K(N^(ε)mPEG5.000-propionyl) desB27 B28E B29R desB30 humaninsulin; 192 A22K(N^(ε)mPEG10.000-propionyl) desB27 B28E B29R desB30human insulin; 193 A22K(N^(ε)mPEG20.000-propionyl) desB27 B28E B29RdesB30 human insulin; 194 A22K(N^(ε)mPEG40.000-propionyl) desB27 B28EB29R desB30 human insulin; 195 A22K(N^(ε)mdPEG₁₂-propionyl) desB27 B28EB29R desB30 human insulin; 196 A22K(N^(ε)mdPEG₂₄-propionyl) desB27 B28EB29R desB30 human insulin; 197 A22K(N^(ε)(mdPEG₁₂)₃-dPEG₄-yl) desB27B28E B29R desB30 human insulin; 198 B28E B29QB31K(N^(ε)mPEG750-propionyl) human insulin; 199 B28E B29QB31K(N^(ε)mPEG2.000-propionyl) human insulin; 200 B28E B29QB31K(N^(ε)mPEG5.000-propionyl) human insulin; 201 B28E B29QB31K(N^(ε)mPEG10.000-propionyl) human insulin; 202 B28E B29QB31K(N^(ε)mPEG20.000-propionyl) human insulin; 203 B28E B29QB31K(N^(ε)mPEG40.000-propionyl) human insulin; 204 B28E B29QB31K(N^(ε)mdPEG₁₂-propionyl) human insulin; 205 B28E B29QB31K(N^(ε)mdPEG₂₄-propionyl) human insulin; 206 B28E B29QB31K(N^(ε)(mdPEG₁₂)₃-dPEG₄-yl) human insulin; 207 desB27 B28E B29QB31K(N^(ε)mPEG750-propionyl) human insulin; 208 desB27 B28E B29QB31K(N^(ε)mPEG2.000-propionyl) human insulin; 209 desB27 B28E B29QB31K(N^(ε)mPEG5.000-propionyl) human insulin; 210 desB27 B28E B29QB31K(N^(ε)mPEG10.000-propionyl) human insulin; 211 desB27 B28E B29QB31K(N^(ε)mPEG20.000-propionyl) human insulin; 212 desB27 B28E B29QB31K(N^(ε)mPEG40.000-propionyl) human insulin; 213 desB27 B28E B29QB31K(N^(ε)mdPEG₁₂-propionyl) human insulin; 214 desB27 B28E B29QB31K(N^(ε)mdPEG₂₄-propionyl) human insulin; 215 desB27 B28E B29QB31K(N^(ε)(mdPEG₁₂)₃-dPEG₄-yl) human insulin; 216A22K(N^(ε)mPEG750-propionyl) B28R desB29 desB30 human insulin; 217A22K(N^(ε)mPEG2.000-propionyl) B28R desB29 desB30 human insulin; 218A22K(N^(ε)mPEG5.000-propionyl) B28R desB29 desB30 human insulin; 219A22K(N^(ε)mPEG10.000-propionyl) B28R desB29 desB30 human insulin; 220A22K(N^(ε)mPEG20.000-propionyl) B28R desB29 desB30 human insulin; 221A22K(N^(ε)mPEG40.000-propionyl) B28R desB29 desB30 human insulin; 222A22K(N^(ε)mdPEG₁₂-propionyl) B28R desB29 desB30 human insulin; 223A22K(N^(ε)mdPEG₂₄-propionyl) B28R desB29 desB30 human insulin; 224A22K(N^(ε)(mdPEG₁₂)₃-dPEG₄-yl) B28R desB29 desB30 human insulin; 225B28R B29P B31K(N^(ε)mPEG750-propionyl) human insulin; 226 B28R B29PB31K(N^(ε)mPEG2.000-propionyl) human insulin; 227 B28R B29PB31K(N^(ε)mPEG5.000-propionyl) human insulin; 228 B28R B29PB31K(N^(ε)mPEG10.000-propionyl) human insulin; 229 B28R B29PB31K(N^(ε)mPEG20.000-propionyl) human insulin; 230 B28R B29PB31K(N^(ε)mPEG40.000-propionyl) human insulin; 231 B28R B29PB31K(N^(ε)mdPEG₁₂-propionyl) human insulin; 232 B28R B29PB31K(N^(ε)mdPEG₂₄-propionyl) human insulin; 233 B28R B29PB31K(N^(ε)(mdPEG₁₂)₃-dPEG₄-yl) human insulin; 234A22K(N^(ε)mPEG750-propionyl) B3Q B28E B29R desB30 human insulin; 235A22K(N^(ε)mPEG2.000-propionyl) B3Q B28E B29R desB30 human insulin; 236A22K(N^(ε)mPEG5.000-propionyl) B3Q B28E B29R desB30 human insulin; 237A22K(N^(ε)mPEG10.000-propionyl) B3Q B28E B29R desB30 human insulin; 238A22K(N^(ε)mPEG20.000-propionyl) B3Q B28E B29R desB30 human insulin; 239A22K(N^(ε)mPEG40.000-propionyl) B3Q B28E B29R desB30 human insulin; 240A22K(N^(ε)mdPEG₁₂-propionyl) B3Q B28E B29R desB30 human insulin; 241A22K(N^(ε)mdPEG₂₄-propionyl) B3Q B28E B29R desB30 human insulin; 242A22K(N^(ε)(mdPEG₁₂)₃-dPEG₄-yl) B3Q B28E B29R desB30 human insulin; 243A21G B3Q B28E B29Q B31K(N^(ε)mPEG750-propionyl) human insulin; 244 A21GB3Q B28E B29Q B31K(N^(ε)mPEG2.000-propionyl) human insulin; 245 A21G B3QB28E B29Q B31K(N^(ε)mPEG5.000-propionyl) human insulin; 246 A21G B3QB28E B29Q B31K(N^(ε)mPEG10.000-propionyl) human insulin; 247 A21G B3QB28E B29Q B31K(N^(ε)mPEG20.000-propionyl) human insulin; 248 A21G B3QB28E B29Q B31K(N^(ε)mPEG40.000-propionyl) human insulin; 249 A21G B3QB28E B29Q B31K(N^(ε)mdPEG₁₂-propionyl) human insulin; 250 A21G B3Q B28EB29Q B31K(N^(ε)mdPEG₂₄-propionyl) human insulin; 251 A21G B3Q B28E B29QB31K(N^(ε)(mdPEG₁₂)₃-dPEG₄-yl) human insulin; 252A22K(N^(ε)mPEG750-propionyl) B13Q B29R desB30 human insulin; 253A22K(N^(ε)mPEG2.000-propionyl) B13Q B29R desB30 human insulin; 254A22K(N^(ε)mPEG5.000-propionyl) B13Q B29R desB30 human insulin; 255A22K(N^(ε)mPEG10.000-propionyl) B13Q B29R desB30 human insulin; 256A22K(N^(ε)mPEG20.000-propionyl) B13Q B29R desB30 human insulin; 257A22K(N^(ε)mPEG40.000-propionyl) B13Q B29R desB30 human insulin; 258A22K(N^(ε)mdPEG₁₂-propionyl) B13Q B29R desB30 human insulin; 259A22K(N^(ε)mdPEG₂₄-propionyl) B13Q B29R desB30 human insulin; 260A22K(N^(ε)((mdPEG₁₂)₃-dPEG₄-yl) B13Q B29R desB30 human insulin; 261A22K(N^(ε)mPEG750-propionyl) desB1 B29R desB30 human insulin; 262A22K(N^(ε)mPEG2.000-propionyl) desB1 B29R desB30 human insulin; 263A22K(N^(ε)mPEG5.000-propionyl) desB1 B29R desB30 human insulin; 264A22K(N^(ε)mPEG10.000-propionyl) desB1 B29R desB30 human insulin; 265A22K(N^(ε)mPEG20.000-propionyl) desB1 B29R desB30 human insulin; 266A22K(N^(ε)mPEG40.000-propionyl) desB1 B29R desB30 human insulin; 267A22K(N^(ε)mdPEG₁₂-propionyl) desB1 B29R desB30 human insulin; 268A22K(N^(ε)mdPEG₂₄-propionyl) desB1 B29R desB30 human insulin; 269A22K(N^(ε)((mdPEG₁₂)₃-dPEG₄-yl) desB1 B29R desB30 human insulin; 270A14E A22K(N^(ε)mPEG750-propionyl) B25H desB30 human insulin; 271 A14EA22K(N^(ε)mPEG2.000-propionyl) B25H desB30 human insulin; 272 A14EA22K(N^(ε)mPEG5.000-propionyl) B25H desB30 human insulin; 273 A14EA22K(N^(ε)mPEG10.000-propionyl) B25H desB30 human insulin; 274 A14EA22K(N^(ε)mPEG20.000-propionyl) B25H desB30 human insulin; 275 A14EA22K(N^(ε)mPEG40.000-propionyl) B25H desB30 human insulin; 276 A14EA22K(N^(ε)mdPEG₁₂-propionyl) B25H desB30 human insulin; 277 A14EA22K(N^(ε)mdPEG₂₄-propionyl) B25H desB30 human insulin; 278 A14EA22K(N^(ε)(mdPEG₁₂)₃-dPEG₄-yl) B25H desB30 human insulin; 279 A14E B25HB29Q B31K(N^(ε)mPEG750-propionyl) human insulin; 280 A14E B25H B29QB31K(N^(ε)mPEG2.000-propionyl) human insulin; 281 A14E B25H B29QB31K(N^(ε)mPEG5.000-propionyl) human insulin; 282 A14E B25H B29QB31K(N^(ε)mPEG10.000-propionyl) human insulin; 283 A14E B25H B29QB31K(N^(ε)mPEG20.000-propionyl) human insulin; 284 A14E B25H B29QB31K(N^(ε)mPEG40.000-propionyl) human insulin; 285 A14E B25H B29QB31K(N^(ε)mdPEG₁₂-propionyl) human insulin; 286 A14E B25H B29QB31K(N^(ε)mdPEG₂₄-propionyl) human insulin; 287 A14E B25H B29QB31K(N^(ε)(mdPEG₁₂)₃-dPEG₄-yl) human insulin; 288 A13EA22K(N^(ε)mPEG750-propionyl) B25H desB30 human insulin; 289 A13EA22K(N^(ε)mPEG2.000-propionyl) B25H desB30 human insulin; 290 A13EA22K(N^(ε)mPEG5.000-propionyl) B25H desB30 human insulin; 291 A13EA22K(N^(ε)mPEG10.000-propionyl) B25H desB30 human insulin; 292 A13EA22K(N^(ε)mPEG20.000-propionyl) B25H desB30 human insulin; 293 A13EA22K(N^(ε)mPEG40.000-propionyl) B25H desB30 human insulin; 294 A13EA22K(N^(ε)mdPEG₁₂-propionyl) B25H desB30 human insulin; 295 A13EA22K(N^(ε)mdPEG₂₄-propionyl) B25H desB30 human insulin; 296 A13EA22K(N^(ε)(mdPEG₁₂)₃-dPEG₄-yl) B25H desB30 human insulin; 297 A21QA22K(N^(ε)mPEG750-propionyl) B29R desB30 human insulin; 298 A21QA22K(N^(ε)mPEG2.000-propionyl) B29R desB30 human insulin; 299 A21QA22K(N^(ε)mPEG5.000-propionyl) B29R desB30 human insulin; 300 A21QA22K(N^(ε)mPEG10.000-propionyl) B29R desB30 human insulin; 301 A21QA22K(N^(ε)mPEG20.000-propionyl) B29R desB30 human insulin; 302 A21QA22K(N^(ε)mPEG40.000-propionyl) B29R desB30 human insulin; 303 A21QA22K(N^(ε)mdPEG₁₂-propionyl) B29R desB30 human insulin; 304 A21QA22K(N^(ε)mdPEG₂₄-propionyl) B29R desB30 human insulin; 305 A21QA22K((mdPEG₁₂)₃-dPEG₄-yl) B29R desB30 human insulin; 306 A21Q A22GA23K(N^(ε)mPEG750-propionyl) B29R desB30 human insulin; 307 A21Q A22GA23K(N^(ε)mPEG2.000-propionyl) B29R desB30 human insulin; 308 A21Q A22GA23K(N^(ε)mPEG5.000-propionyl) B29R desB30 human insulin; 309 A21Q A22GA23K(N^(ε)mPEG10.000-propionyl) B29R desB30 human insulin; 310 A21Q A22GA23K(N^(ε)mPEG20.000-propionyl) B29R desB30 human insulin; 311 A21Q A22GA23K(N^(ε)mPEG40.000-propionyl) B29R desB30 human insulin; 312 A21Q A22GA23K(N^(ε)mdPEG₁₂-propionyl) B29R desB30 human insulin; 313 A21Q A22GA23K(N^(ε)mdPEG₂₄-propionyl) B29R desB30 human insulin; 314 A21Q A22GA23K((mdPEG₁₂)₃-dPEG₄-yl) B29R desB30 human insulin; 315 A21Q A22G A23GA24K(N^(ε)mPEG750-propionyl) B29R desB30 human insulin; 316 A21Q A22GA23G A24K(N^(ε)mPEG2.000-propionyl) B29R desB30 human insulin; 317 A21QA22G A23G A24K(N^(ε)mPEG5.000-propionyl) B29R desB30 human insulin; 318A21Q A22G A23G A24K(N^(ε)mPEG10.000-propionyl) B29R desB30 humaninsulin; 319 A21Q A22G A23G A24K(N^(ε)mPEG20.000-propionyl) B29R desB30human insulin; 320 A21Q A22G A23G A24K(N^(ε)mPEG40.000-propionyl) B29RdesB30 human insulin; 321 A21Q A22G A23G A24K(N^(ε)mdPEG₁₂-propionyl)B29R desB30 human insulin; 322 A21Q A22G A23GA24K(N^(ε)mdPEG₂₄-propionyl) B29R desB30 human insulin; 323 A21Q A22GA23G A24K((mdPEG₁₂)₃-dPEG₄-yl) B29R desB30 human insulin; 324 A21Q A22GA23G A24G A25K(N^(ε)mPEG750-propionyl) B29R desB30 human insulin; 325A21Q A22G A23G A24G A25K(N^(ε)mPEG2.000-propionyl) B29R desB30 humaninsulin; 326 A21Q A22G A23G A24G A25K(N^(ε)mPEG5.000-propionyl) B29RdesB30 human insulin; 327 A21Q A22G A23G A24GA25K(N^(ε)mPEG10.000-propionyl) B29R desB30 human insulin; 328 A21Q A22GA23G A24G A25K(N^(ε)mPEG20.000-propionyl) B29R desB30 human insulin; 329A21Q A22G A23G A24G A25K(N^(ε)mPEG40.000-propionyl) B29R desB30 humaninsulin; 330 A21Q A22G A23G A24G A25K(N^(ε)mdPEG₁₂-propionyl) B29RdesB30 human insulin; 331 A21Q A22G A23G A24GA25K(N^(ε)mdPEG₂₄-propionyl) B29R desB30 human insulin; 332 A21Q A22GA23G A24G A25K((mdPEG₁₂)₃-dPEG₄-yl) B29R desB30 human insulin; 333 A21AA22K(N^(ε)mPEG750-propionyl) B29R desB30 human insulin; 334 A21AA22K(N^(ε)mPEG2.000-propionyl) B29R desB30 human insulin; 335 A21AA22K(N^(ε)mPEG5.000-propionyl) B29R desB30 human insulin; 336 A21AA22K(N^(ε)mPEG10.000-propionyl) B29R desB30 human insulin; 337 A21AA22K(N^(ε)mPEG20.000-propionyl) B29R desB30 human insulin; 338 A21AA22K(N^(ε)mPEG40.000-propionyl) B29R desB30 human insulin; 339 A21AA22K(N^(ε)mdPEG₁₂-propionyl) B29R desB30 human insulin; 340 A21AA22K(N^(ε)mdPEG₂₄-propionyl) B29R desB30 human insulin; 341 A21AA22K((mdPEG₁₂)₃-dPEG₄-yl) B29R desB30 human insulin; 342 A21A A22GA23K(N^(ε)mPEG750-propionyl) B29R desB30 human insulin; 343 A21A A22GA23K(N^(ε)mPEG2.000-propionyl) B29R desB30 human insulin; 344 A21A A22GA23K(N^(ε)mPEG5.000-propionyl) B29R desB30 human insulin; 345 A21A A22GA23K(N^(ε)mPEG10.000-propionyl) B29R desB30 human insulin; 346 A21A A22GA23K(N^(ε)mPEG20.000-propionyl) B29R desB30 human insulin; 347 A21A A22GA23K(N^(ε)mPEG40.000-propionyl) B29R desB30 human insulin; 348 A21A A22GA23K(N^(ε)mdPEG₁₂-propionyl) B29R desB30 human insulin; 349 A21A A22GA23K(N^(ε)mdPEG₂₄-propionyl) B29R desB30 human insulin; 350 A21A A22GA23K((mdPEG₁₂)₃-dPEG₄-yl) B29R desB30 human insulin; 351 A21A A22G A23GA24K(N^(ε)mPEG750-propionyl) B29R desB30 human insulin; 352 A21A A22GA23G A24K(N^(ε)mPEG2.000-propionyl) B29R desB30 human insulin; 353 A21AA22G A23G A24K(N^(ε)mPEG5.000-propionyl) B29R desB30 human insulin; 354A21A A22G A23G A24K(N^(ε)mPEG10.000-propionyl) B29R desB30 humaninsulin; 355 A21A A22G A23G A24K(N^(ε)mPEG20.000-propionyl) B29R desB30human insulin; 356 A21A A22G A23G A24K(N^(ε)mPEG40.000-propionyl) B29RdesB30 human insulin; 357 A21A A22G A23G A24K(N^(ε)mdPEG₁₂-propionyl)B29R desB30 human insulin; 358 A21A A22G A23GA24K(N^(ε)mdPEG₂₄-propionyl) B29R desB30 human insulin; 359 A21A A22GA23G A24K((mdPEG₁₂)₃-dPEG₄-yl) B29R desB30 human insulin; 360 A21A A22GA23G A24G A25K(N^(ε)mPEG750-propionyl) B29R desB30 human insulin; 361A21A A22G A23G A24G A25K(N^(ε)mPEG2.000-propionyl) B29R desB30 humaninsulin; 362 A21A A22G A23G A24G A25K(N^(ε)mPEG5.000-propionyl) B29RdesB30 human insulin; 363 A21A A22G A23G A24GA25K(N^(ε)mPEG10.000-propionyl) B29R desB30 human insulin; 364 A21A A22GA23G A24G A25K(N^(ε)mPEG20.000-propionyl) B29R desB30 human insulin; 365A21A A22G A23G A24G A25K(N^(ε)mPEG40.000-propionyl) B29R desB30 humaninsulin; 366 A21A A22G A23G A24G A25K(N^(ε)mdPEG₁₂-propionyl) B29RdesB30 human insulin; 367 A21A A22G A23G A24GA25K(N^(ε)mdPEG₂₄-propionyl) B29R desB30 human insulin; 368 A21A A22GA23G A24G A25K((mdPEG₁₂)₃-dPEG₄-yl) B29R desB30 human insulin; 369 A21GA22K(N^(ε)mPEG750-propionyl) B29R desB30 human insulin; 370 A21GA22K(N^(ε)mPEG2.000-propionyl) B29R desB30 human insulin; 371 A21GA22K(N^(ε)mPEG5.000-propionyl) B29R desB30 human insulin; 372 A21GA22K(N^(ε)mPEG10.000-propionyl) B29R desB30 human insulin; 373 A21GA22K(N^(ε)mPEG20.000-propionyl) B29R desB30 human insulin; 374 A21GA22K(N^(ε)mPEG40.000-propionyl) B29R desB30 human insulin; 375 A21GA22K(N^(ε)mdPEG₁₂-propionyl) B29R desB30 human insulin; 376 A21GA22K(N^(ε)mdPEG₂₄-propionyl) B29R desB30 human insulin; 377 A21GA22K((mdPEG₁₂)₃-dPEG₄-yl) B29R desB30 human insulin; 378 A21G A22GA23K(N^(ε)mPEG750-propionyl) B29R desB30 human insulin; 379 A21G A22GA23K(N^(ε)mPEG2.000-propionyl) B29R desB30 human insulin; 380 A21G A22GA23K(N^(ε)mPEG5.000-propionyl) B29R desB30 human insulin; 381 A21G A22GA23K(N^(ε)mPEG10.000-propionyl) B29R desB30 human insulin; 382 A21G A22GA23K(N^(ε)mPEG20.000-propionyl) B29R desB30 human insulin; 383 A21G A22GA23K(N^(ε)mPEG40.000-propionyl) B29R desB30 human insulin; 384 A21G A22GA23K(N^(ε)mdPEG₁₂-propionyl) B29R desB30 human insulin; 385 A21G A22GA23K(N^(ε)mdPEG₂₄-propionyl) B29R desB30 human insulin; 386 A21G A22GA23K((mdPEG₁₂)₃-dPEG₄-yl) B29R desB30 human insulin; 387 A21G A22G A23GA24K(N^(ε)mPEG750-propionyl) B29R desB30 human insulin; 388 A21G A22GA23G A24K(N^(ε)mPEG2.000-propionyl) B29R desB30 human insulin; 389 A21GA22G A23G A24K(N^(ε)mPEG5.000-propionyl) B29R desB30 human insulin; 390A21G A22G A23G A24K(N^(ε)mPEG10.000-propionyl) B29R desB30 humaninsulin; 391 A21G A22G A23G A24K(N^(ε)mPEG20.000-propionyl) B29R desB30human insulin; 392 A21G A22G A23G A24K(N^(ε)mPEG40.000-propionyl) B29RdesB30 human insulin; 393 A21G A22G A23G A24K(N^(ε)mdPEG₁₂-propionyl)B29R desB30 human insulin; 394 A21G A22G A23GA24K(N^(ε)mdPEG₂₄-propionyl) B29R desB30 human insulin; 395 A21G A22GA23G A24K((mdPEG₁₂)₃-dPEG₄-yl) B29R desB30 human insulin; 396 A21G A22GA23G A24G A25K(N^(ε)mPEG750-propionyl) B29R desB30 human insulin; 397A21G A22G A23G A24G A25K(N^(ε)mPEG2.000-propionyl) B29R desB30 humaninsulin; 398 A21G A22G A23G A24G A25K(N^(ε)mPEG5.000-propionyl) B29RdesB30 human insulin; 399 A21G A22G A23G A24GA25K(N^(ε)mPEG10.000-propionyl) B29R desB30 human insulin; 400 A21G A22GA23G A24G A25K(N^(ε)mPEG20.000-propionyl) B29R desB30 human insulin; 401A21G A22G A23G A24G A25K(N^(ε)mPEG40.000-propionyl) B29R desB30 humaninsulin; 402 A21G A22G A23G A24G A25K(N^(ε)mdPEG₁₂-propionyl) B29RdesB30 human insulin; 403 A21G A22G A23G A24GA25K(N^(ε)mdPEG₂₄-propionyl) B29R desB30 human insulin; 404 A21G A22GA23G A24G A25K((mdPEG₁₂)₃-dPEG₄-yl) B29R desB30 human insulin; 405 A21QA22K(N^(ε)mPEG750-propionyl) B3Q B29R desB30 human insulin; 406 A21QA22K(N^(ε)mPEG2.000-propionyl) B3Q B29R desB30 human insulin; 407 A21QA22K(N^(ε)mPEG5.000-propionyl) B3Q B29R desB30 human insulin; 408 A21QA22K(N^(ε)mPEG10.000-propionyl) B3Q B29R desB30 human insulin; 409 A21QA22K(N^(ε)mPEG20.000-propionyl) B3Q B29R desB30 human insulin; 410 A21QA22K(N^(ε)mPEG40.000-propionyl) B3Q B29R desB30 human insulin; 411 A21QA22K(N^(ε)mdPEG₁₂-propionyl) B3Q B29R desB30 human insulin; 412 A21QA22K(N^(ε)mdPEG₂₄-propionyl) B3Q B29R desB30 human insulin; 413 A21QA22K((mdPEG₁₂)₃-dPEG₄-yl) B3Q B29R desB30 human insulin; 414 A21GA22K(N^(ε)mPEG750-propionyl) B3Q B29R desB30 human insulin; 415 A21GA22K(N^(ε)mPEG2.000-propionyl) B3Q B29R desB30 human insulin; 416 A21GA22K(N^(ε)mPEG5.000-propionyl) B3Q B29R desB30 human insulin; 417 A21GA22K(N^(ε)mPEG10.000-propionyl) B3Q B29R desB30 human insulin; 418 A21GA22K(N^(ε)mPEG20.000-propionyl) B3Q B29R desB30 human insulin; 419 A21GA22K(N^(ε)mPEG40.000-propionyl) B3Q B29R desB30 human insulin; 420 A21GA22K(N^(ε)mdPEG₁₂-propionyl) B3Q B29R desB30 human insulin; 421 A21GA22K(N^(ε)mdPEG₂₄-propionyl) B3Q B29R desB30 human insulin; 422 A21GA22K((mdPEG₁₂)₃-dPEG₄-yl) B3Q B29R desB30 human insulin; 423 A21AA22K(N^(ε)mPEG750-propionyl) B3Q B29R desB30 human insulin; 424 A21AA22K(N^(ε)mPEG2.000-propionyl) B3Q B29R desB30 human insulin; 425 A21AA22K(N^(ε)mPEG5.000-propionyl) B3Q B29R desB30 human insulin; 426 A21AA22K(N^(ε)mPEG10.000-propionyl) B3Q B29R desB30 human insulin; 427 A21AA22K(N^(ε)mPEG20.000-propionyl) B3Q B29R desB30 human insulin; 428 A21AA22K(N^(ε)mPEG40.000-propionyl) B3Q B29R desB30 human insulin; 429 A21AA22K(N^(ε)mdPEG₁₂-propionyl) B3Q B29R desB30 human insulin; 430 A21AA22K(N^(ε)mdPEG₂₄-propionyl) B3Q B29R desB30 human insulin; 431 A21AA22K((mdPEG₁₂)₃-dPEG₄-yl) B3Q B29R desB30 human insulin.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 and FIG. 2 is the rat intratracheal drop instillation of theinsulin of example 1 and 2.

FIG. 3 is the rat intratracheal drop instillation of the insulin ofexample 6.

FIG. 4 is the rat intratracheal drop instillation of the insulin ofexample 5.

FIG. 5 is the rat intratracheal drop instillation of the insulin ofexample 16.

FIG. 6 is the rat intratracheal drop instillation of the insulin ofexample 18.

FIG. 7 is the rat intratracheal drop instillation of the insulin ofexample 17.

FIG. 8 is the rat intratracheal drop instillation of the insulin ofexample 19.

FIG. 9 is the rat intratracheal drop instillation of the insulin ofexample 22.

FIG. 10 is the blood glucose profile by pulmonary administration of aspray dried powder of the insulin of examples 1 and 2 to mini-pigs wherethe mean dose delivered was 0.037±0.009 mg/kg.

FIG. 11 is the pharmacokinetic profile by pulmonary administration of aspray dried powder of the insulin of examples 1 and 2 to mini-pigs wherethe mean dose delivered was 0.037±0.009 mg/kg.

1. A PEGylated insulin analogue which, compared with human insulin, has one or more PEG-containing extensions extended from the A1, B1, A21 and/or B30 position(s), said extension(s) consist(s) of amino acid residue(s) and wherein the PEG moiety, via a linker, is attached to an amino acid residue in the extension and with the proviso that, preferably, the parent insulin analogue contains only one lysine residue.
 2. The PEGylated insulin analogue according to claim 1, wherein only one of the extensions carries a PEG moiety and, preferably, there is only one extension.
 3. The PEGylated insulin analogue according to claim 1, wherein the extension carrying a PEG moiety is situated in a position C-terminally to the A21 position.
 4. The PEGylated insulin analogue according to claim 1, wherein the extension carrying a PEG moiety is situated in a position C-terminally to the B30 position.
 5. The PEGylated insulin analogue according to claim 1, wherein the parent insulin analogue deviates from human insulin in one or more of the following extensions: G in position A-3, G in position A-2, K or R in position A-1, G or K in position A22, G or K in position A23, G or K in position A24, K in position A25, and K in position B31 and, compared with human insulin, there is, optionally, up to 12, preferably up to 8, more preferred up to 4, more mutations among deletion, substitution and addition of an amino acid residue and, preferably, there are no further mutations in said insulin analogue and with the proviso that, preferably, the parent insulin analogue contains only one lysine residue.
 6. The PEGylated insulin analogue according to claim 1, wherein the extension consists of one or more of the following formulae wherein the PEG moiety is attached to side chain(s) of lysine or cysteine residue(s) when present or to the N-terminal amino group(s) (or both): -AA_(x1)K (for C-terminal extensions), wherein X1 is 0, 1, 2, 3, 4, 5, 6, 7, or 8 (and wherein K is lysine), K-AA_(x2)- (for N-terminal extensions), wherein x2 is 0, 1, 2, 3, 4, 5, 6, 7, or 8 (and wherein K is lysine), -AA_(x3)C (for C-terminal extensions), wherein x3 is 0, 1, 2, 3, 4, 5, 6, 7, or 8 (and wherein C is cysteine), C-AA_(x4)- (for N-terminal extensions), wherein x4 is 0, 1, 2, 3, 4, 5, 6, 7, or 8 (and wherein C is cysteine), AA_(x5)-R_(y)- (for N-terminal extensions), wherein x5 is 0, 1, 2, 3, 4, 5, 6, 7, or 8, and y is 0 or 1 (and wherein R is arginine), and wherein AA is a residue of a peptide chain wherein each of the amino acid residues are the same or different and each is any codable amino acid except Lys and Cys.
 7. The PEGylated insulin analogue according to claim 1, wherein AA is a peptide residue consisting of amino acid residues of glycine, alanine or glutamine.
 8. The PEGylated insulin analogue according to claim 7, wherein AA is a residue of glycine.
 9. The PEGylated insulin analogue according to claim 7, wherein AA is a residue of alanine.
 10. The PEGylated insulin analogue according to claim 7, wherein AA is a residue of glutamine.
 11. The PEGylated insulin analogue according to claim 1 wherein the parent insulin, optionally contains one or more of the following mutations: A14E/D, A18Q, A21G/A/Q, desB1, B1G/Q, B3Q/S/T, B13Q, desB25, B25H, desB27, B28D/E/R, desB29, B29P/Q/R or desB30.
 12. The PEGylated insulin analogue according to claim 1, wherein the parent insulin analogue deviates from human insulin in having A22K, B29R and desB30 and, preferably, there are no further mutations in said insulin analogue.
 13. The PEGylated insulin analogue according to claim 1, wherein the parent insulin analogue deviates from human insulin in having A22G, A23K, B29R and desB30 and, preferably, there are no further mutations in said insulin analogue.
 14. The A PEGylated insulin analogue according to claim 1, comprising the moiety —(OCH₂CH₂)_(n)—, wherein n is in integer in the range from 2 to about 1000, preferably from 2 to about 500, preferably from 2 to about 250, preferably from 2 to about 125, preferably from 2 to about 50, and preferably from 2 to about
 25. 15. The PEGylated insulin analogue according to claim 1, which is one of the following PEGylated insulin analogues: a) a PEGylated insulin analogue wherein the parent insulin analogue deviates from human insulin in having A22G, A23G, A24K, B29R and desB30 and, preferably, there are no further mutations in said insulin analogue, b) a PEGylated insulin analogue wherein the parent insulin analogue deviates from human insulin in having A22G, A23G, A24G, A25K, B29R and desB30 and, preferably, there are no further mutations in said insulin analogue, c) a PEGylated insulin analogue wherein the parent insulin analogue deviates from human insulin in having A21Q, A22K, B29R and desB30 and, preferably, there are no further mutations in said insulin analogue, d) a PEGylated insulin analogue wherein the parent insulin analogue deviates from human insulin in having A21Q, A22G, A23K, B29R and desB30 and, preferably, there are no further mutations in said insulin analogue, e) a PEGylated insulin analogue wherein the parent insulin analogue deviates from human insulin in having A21A, A22K, B29R and desB30 and, preferably, there are no further mutations in said insulin analogue, f) a PEGylated insulin analogue wherein the parent insulin analogue deviates from human insulin in having A21G, A22K, B29R and desB30 and, preferably, there are no further mutations in said insulin analogue, g) a PEGylated insulin analogue comprising a group of the general formula -Q¹-(OCH₂CH₂)_(n)—R¹, wherein Q¹ is a linker connecting the polyethylene glycol moiety to an α- or γ-NH-group of an amino acid in the extension, preferably via an amide or a carbamate bond, n is an integer in the range from 2 to about 1000, and R¹ is alkoxy or hydroxyl, preferably methoxy, and h) a PEGylated insulin analogue wherein the parent insulin analogue deviates from human insulin in having A14E, A22K, B25H, B29R and desB30 and, preferably, there are no further mutations in said insulin analogue. 